Abstract

Background

The effect of neonatal anesthesia and pain on the developing brain is of considerable clinical importance, but few studies have evaluated noxious surgical input to the infant brain under anesthesia. Herein, the authors tested the effect of increasing isoflurane concentration on spontaneous and evoked nociceptive activity in the somatosensory cortex of rats at different postnatal ages.

Methods

Intracortical extracellular field potentials evoked by hind paw C-fiber electrical stimulation were recorded in the rat somatosensory cortex at postnatal day (P) 7, P14, P21, and P30 during isoflurane anesthesia (n = 7 per group). The amplitudes of evoked potentials and the energies of evoked oscillations (1 to 100 Hz over 3 s) were measured after equilibration at 1.5% isoflurane and during step increases in inspired isoflurane. Responses during and after plantar hind paw incision were compared at P7 and P30 (n = 6 per group).

Results

At P7, cortical activity was silent at 1.5% isoflurane but noxious-evoked potentials decreased only gradually in amplitude and energy with step increases in isoflurane. The resistance of noxious-evoked potentials to isoflurane at P7 was significantly enhanced after surgical hind paw incision (69 ± 16% vs. 6 ± 1% in nonincised animals at maximum inspired isoflurane). This resistance was age dependent; at P14 to P30, noxious-evoked responses decreased sharply with increasing isoflurane (step 3 [4%] P7: 50 ± 9%, P30: 4 ± 1% of baseline). Hind paw incision at P30 sensitized noxious-evoked potentials, but this was suppressed by higher isoflurane concentrations.

Conclusions

Despite suppression of spontaneous activity, cortical-evoked potentials are more resistant to isoflurane in young rats and are further sensitized by surgical injury.

Abstract

Extracellular somatosensory cortex field potentials evoked by hind paw C-fiber electrical stimulation were resistant to isoflurane compared with spontaneous activity in neonatal rat. Surgical hind paw incision enhanced the resistance of noxious-evoked responses to isoflurane, an effect that declined with age, indicating critical age-dependent differences in anesthetic suppression of cortical nociceptive activity.

What We Already Know about This Topic
  • Considerable evidence indicates that neonatal anesthesia and tissue injury have long-term consequences on somatosensory and nociceptive systems

  • The anesthetic sensitivity of noxious cutaneous–evoked activity in the neonatal somatosensory cortex, with or without surgical trauma, is unknown

What This Article Tells Us That Is New
  • Extracellular somatosensory cortex field potentials evoked by hind paw C-fiber electrical stimulation were resistant to isoflurane compared with spontaneous activity in neonatal rat

  • Surgical hind paw incision enhanced the resistance of noxious-evoked responses to isoflurane, an effect that declined with age, indicating critical age-dependent differences in anesthetic suppression of cortical nociceptive activity

AN optimal level of neonatal anesthesia achieves both hypnosis and antinociception while maintaining physiologic stability and minimizing potential neurotoxicity.1,2  As both anesthesia and uncontrolled pain may alter cortical activity and impair neurodevelopmental outcomes,3–5  the impact of anesthetic agents on both spontaneous and noxious-evoked neural activity in the developing brain requires further evaluation. An important aspect of neonatal anesthesia research is the effect of nociceptive sensory input on activity within cortical sensory circuits and the degree to which central nociceptive activity is modulated by anesthesia and analgesia. Both animal and clinical evidence point to long-term consequences of early life procedural and surgical tissue injury on somatosensory and nociceptive systems,6,7  highlighting the need to consider the impact of postnatal age on changes in both spontaneous and noxious-evoked cortical activity during surgery and anesthesia.

Extracellular field recording, including electroencephalogram, electrocorticogram, and local field potentials (intracortical activity) are commonly used to monitor ongoing spontaneous brain activity and levels of anesthesia in human and rodent neonates but are also used to record specific potentials evoked by a sensory stimulus. Somatosensory potentials evoked by experimental noxious cutaneous stimulation8–11  are commonly used to measure pain activity in the adult human and rodent brain.12  Specific nociceptive potentials are also evoked by single, clinically required skin breaking procedures in the human infant brain13,14  and have been used in this age group to measure the postnatal development of cortical pain processing.10  In adult humans and rodents, nociceptive-evoked potential amplitudes decrease with increasing concentrations of isoflurane,9,15  but, to date, the sensitivity of nociceptive potentials to anesthesia in infants has not been studied. In a study of the whisker barrel cortex of neonatal rats, sensory potentials evoked by whisker deflection persisted at surgical isoflurane levels (1.5 to 2%) that completely suppressed the electroencephalogram and silenced spontaneous neuronal firing.16  This suggests that noxious-evoked potentials and spontaneous intracortical activity might be differentially sensitive to anesthesia in infants. Furthermore, because nociceptive potential amplitudes can be increased by peripheral C-fiber sensitization in both humans and rat cortex,8,11  surgical injury may enhance evoked activity and add to this differential response to anesthesia in infancy.

The primary aim of this experimental laboratory study was to test the impact of increasing isoflurane concentration on the pattern of spontaneous and evoked nociceptive activity in the somatosensory cortex of infant rats undergoing hind paw incision. In addition, we compared the effects of isoflurane on cortical activity at different postnatal ages. We hypothesized that, in infant rats, noxious-evoked brain activity is more resistant than spontaneous brain activity to isoflurane anesthesia and that this difference declines with postnatal age.

Materials and Methods

Animals

All experiments were performed under personal and project licenses approved by the Home Office, London, United Kingdom, under regulations of the UK Animal (Scientific Procedures) Act, 1986. Male Sprague-Dawley rats aged postnatal day (P) 7, P14, P21, and P30 were obtained from the Biological Services Unit, University College London, London, United Kingdom. All animals were from the same colony, bred and maintained in-house, and exposed to the same caging, diet, and handling throughout development. Rats were housed in cages of six age-matched animals (P30) or with the dam and littermates (P7, P14, and P21) under controlled environmental conditions (24° to 25°C; 50 to 60% humidity; 12-h light/dark cycle) with free access to food and water. Animals were randomly picked from litters by hand for recording and alternately assigned to an incision or no incision group. Treatment groups were distributed across multiple litters and/or adult cage groups. At the end of an experiment, the isoflurane was increased to 5% until there was no heart beat and the neck dislocated.

Cortical Recordings

Rats were anaesthetized with 4% isoflurane (Abbot, AbbVie Ltd., United Kingdom) in 100% oxygen through a nose cone and after insertion of a tracheal cannula were mechanically ventilated (Small Animal Ventilator, Bioscience, United Kingdom) with 100% oxygen and isoflurane delivered from a recently calibrated vaporizer (Harvard Apparatus, USA). The adjustments for ventilation of rats across the ages were based on the breath rates and tidal volume. The intermittent positive pressure ventilation was achieved by using a T-type system in conjunction with a small animal lung ventilator. This system affords control of inspired gas mixture (isoflurane concentration and oxygen) inspiratory flow rate, respiratory rate, and peak inspiratory pressure. A simple water manometer placed in the inspiratory limb provided a monitoring and pressure limiting device with visual display. Using an inspiratory flow rate between 1 and 1.5 l/min and adjusting the peak inspiratory pressure between 12 and 15 cm H2O17  allowed the tidal volume to be adjusted according to the size of animals. This mode was confirmed to be adequate in our laboratory by using a transcutaneous combination probe to monitor transcutaneous oxygen and carbon dioxide. Body temperature was maintained with a thermostatically controlled heated blanket, and the electrocardiogram was monitored throughout (NeuroLog, Digitimer, United Kingdom).

During anesthesia with 2.5% isoflurane, animals were placed in a stereotaxic frame, and a craniotomy was performed to expose the surface of the cerebral cortex. A recording electrode (stainless steel, E363/1, Plastic One Inc., USA) was inserted into the primary somatosensory cortex in the somatotopic region for the hind paw. Coordinates for P7 and P14 rats were lateral 2.0 mm from midline and posterior 0.5 mm from the bregma; and coordinates for P21 and P30 rats were lateral 2.5 mm from midline and posterior 1 mm from the bregma.18,19  The reference electrode was placed subcutaneously on the surface of the skull anterior to the bregma, and the ground electrode was placed subcutaneously in the back. The inspired isoflurane concentration was then reduced to 1.5% and allowed to equilibrate for at least 40 min.

Continuous intracortical activity recordings were performed using a NeuroLog NL100 headstage connected to a NL104 amplifier and a NL125 filter (100 Hz). The signal was digitized at 16K Hz using Axon Instruments (Digidata 1400A, Molecular Devices, USA). Data were acquired and stored using a Windows PC–based program, WinEDR v3.3.6 (John Dempster, University of Strathclyde, United Kingdom) for later analysis. The depth of the recording electrode was adjusted to optimize the somatosensory-evoked potential amplitude. At the end of an experiment, the brain was removed and immediately immersed in 4% paraformaldehyde for over 24 h and then transferred to 30% sucrose postfixation solution. Brain sections (40-μm thick thickness) were cut using a micotome (Leica SM2000R, Leica Microsystems (UK) Ltd., United Kingdom) and stained with cresyl violet for histologic location of the electrode track. This procedure verified that recordings were in layer 5 to 6 of the somatosensory cortex (fig. 1). All the data recorded were included in this study, and no animals were excluded.

Fig. 1.

(A) Diagram showing the experimental timelines and recording sites. Animals were initially anesthetized with 2.5 to 4% isoflurane in oxygen for surgical preparation and electrode insertion and then maintained at 1.5% for 40 min to allow equilibration. The electrocardiogram was monitored throughout. Group 1: On postnatal day (P) 7, 14, 21, or 30, continuous intracortical spontaneous activity in the right somatosensory cortex was recorded for 100 s, followed by evoked responses to electrical stimulation of the left hind paw (10 stimuli of 3.2 mA for 500 ms at 10-s intervals). Recordings were repeated at 5-min intervals after step increases in inspired isoflurane concentration: step 1 (2%), step 2 (3%), step 3 (4%), and step 4 (5%). Group 2: In P7 and P30 animals, spontaneous activity was recorded 2 min before, during, and 2 min after left plantar hind paw incision. Ten minutes after incision, spontaneous and electrical activity were recorded during 1.5% and step increases in inspired isoflurane concentration. (B) Recording sites in layer 5/6 of the rat somatosensory cortex. Schematic representation of the location site of intracortical recording in the area of primary somatosensory representing the hind paw (S1HL) in rats aged P7, P14, P21, and P30 (P7 = 12; P14 = 6; P21 = 6; P30 = 12). The locations were determined by post hoc histologic analysis, and recording sites (filled circles) were reconstructed using a standard stereotaxic atlas (Paxinos and Watson18  and Paxinos et al.19 ). L1 = layer 1; L2/3 = layer 2 and 3; L4 = layer 4; L5 = layer 5; L6 = layer 6; WM = white matter.

Fig. 1.

(A) Diagram showing the experimental timelines and recording sites. Animals were initially anesthetized with 2.5 to 4% isoflurane in oxygen for surgical preparation and electrode insertion and then maintained at 1.5% for 40 min to allow equilibration. The electrocardiogram was monitored throughout. Group 1: On postnatal day (P) 7, 14, 21, or 30, continuous intracortical spontaneous activity in the right somatosensory cortex was recorded for 100 s, followed by evoked responses to electrical stimulation of the left hind paw (10 stimuli of 3.2 mA for 500 ms at 10-s intervals). Recordings were repeated at 5-min intervals after step increases in inspired isoflurane concentration: step 1 (2%), step 2 (3%), step 3 (4%), and step 4 (5%). Group 2: In P7 and P30 animals, spontaneous activity was recorded 2 min before, during, and 2 min after left plantar hind paw incision. Ten minutes after incision, spontaneous and electrical activity were recorded during 1.5% and step increases in inspired isoflurane concentration. (B) Recording sites in layer 5/6 of the rat somatosensory cortex. Schematic representation of the location site of intracortical recording in the area of primary somatosensory representing the hind paw (S1HL) in rats aged P7, P14, P21, and P30 (P7 = 12; P14 = 6; P21 = 6; P30 = 12). The locations were determined by post hoc histologic analysis, and recording sites (filled circles) were reconstructed using a standard stereotaxic atlas (Paxinos and Watson18  and Paxinos et al.19 ). L1 = layer 1; L2/3 = layer 2 and 3; L4 = layer 4; L5 = layer 5; L6 = layer 6; WM = white matter.

Electrical Stimulation of the Hind Paw

Two stainless steel pin electrodes were placed subcutaneously 3 to 5 mm apart on the plantar hind paw, contralateral to the cortical recording site. A train of 10 stimuli of 3.2 mA, 500 μs were applied at 10-s interstimulus intervals, using a constant current stimulator (NeuroLog, Digitimer). These stimulation parameters are sufficient to recruit both A- and C-fibers20  (here called “C-fiber” stimulation) and were established in pilot experiments to evoke clear potentials restricted to the somatosensory cortex. At all ages, electrical hind paw stimulation failed to evoke visible hindlimb reflex responses at 1.5% or higher inspired isoflurane concentrations. In a separate group of P7 animals, a train of lower intensity stimuli of 0.32 mA for 50 μs was also applied, sufficient to recruit only Aβ-fibers20  (here called “A-fiber” stimulation), for comparison.

Plantar Hind Paw Incision

Plantar hind paw incision was performed in P7 and P30 animals (n = 6 per age group) after equilibration at 1.5% isoflurane in oxygen. A midline longitudinal incision through the skin and fascia extended from the midpoint of the heel to the proximal border of the first footpad to incise a similar relative length of paw at different ages, and the underlying plantar muscle was elevated and incised.21,22  Skin edges were closed with 5-0 nylon suture (Ethicon, United Kingdom), and the procedure took 2.5 to 3 min. The initial skin incision evoked a brief visible muscle reflex in young animals (P7) but not in older animals (P30).

Experimental Timeline

The experimental timeline is illustrated in figure 1. After surgical preparation and equilibration at 1.5% inspired isoflurane concentration, spontaneous intracortical activity was recorded for 100 s, followed by evoked activity during hind paw electrical stimulation in P7, P14, P21, and P30 rats (fig. 1, Group 1). In pilot studies, and subsequent experimental recordings, it was clear that increasing the inspired isoflurane concentration rapidly altered spontaneous activity. Therefore, the inspired isoflurane concentration was increased at 5-min intervals (step 1 = 2%, step 2 = 3%, step 3 = 4%, and step 4 = 5%), and spontaneous and evoked potentials were measured in the 3- to 5-min period after each step. The step terminology was adopted because this frequency of change would not have allowed sufficient time for equilibration at each inspired concentration. However, this protocol allowed us to assess the response to increasing isoflurane exposure without the cardiovascular compromise associated with more prolonged exposure to high concentrations.

In additional groups at P7 or P30, a plantar hind paw incision was performed after equilibration at 1.5% isoflurane (fig. 1, Group 2). In these experiments, spontaneous activity was measured (sampled from a 100-s epoch) in the 2-min period before, during, and 2 min after plantar hind paw incision with inspired isoflurane maintained at 1.5%. Ten minutes after plantar hind paw incision, recordings were performed during step increases in inspired isoflurane concentration as described for group 1.

Electrocardiography confirmed that the heart rate remained stable throughout the recording periods at 1.5% isoflurane in both groups. At all ages, the heart rate was reduced by step 4. When expressed as a percentage change from baseline (as heart rate differs with postnatal age in rodents), the degree of change in heart rate was not significantly different at P7 and P30 (P7: 81 ± 3% vs. P30: 88 ± 3%, P = 0.157, Student’s t test; data not shown).

Statistical Analysis

Sample sizes (n = 7 animals for each age) were based on previously published group effects of volatile anesthesia on cortical activity. Statistically significant dose-dependent effects of isoflurane on evoked responses have been reported in the somatosensory cortex of adult rats (n = 5)9  and in the barrel cortex of neonatal (P2 to P7) rats (n = 6).16 

Ongoing spontaneous intracortical activity was analyzed in 100-s epochs at each isoflurane step and before, during, and after plantar hind paw incision. Frequency domain analysis was performed by fast Fourier transformation (spectrum type: one sided) using the “Welch Window.”23  Intracortical responses were converted from the time domain to the frequency domain, and the Fourier transform components amplitude (millivolts per Hertz) was computed using OriginPro 9 (OriginLab Corporation, USA). Brain waves have been categorized into five frequency bands: delta (δ = 2 to 4 Hz), theta (θ = 4 to 8 Hz), alpha (α = 8 to 12 Hz), beta (β = 13 to 30 Hz), and gamma (γ more than 30 Hz).24  Network oscillations with a range of frequencies are thought to control the flow of information through anatomical pathways and communicate among local networks. The changing patterns of network oscillation are tightly correlated with behavior or features of sensory stimuli.25 

Individual noxious-evoked potentials were averaged (10 stimuli per animal), and peak amplitudes were computed for each animal. Data are given as grand mean ± SEM (number of stimuli × number of animals). In addition, evoked potentials at steps 2 to 5 were normalized to baseline (1.5% of inspired isoflurane) and presented as % of baseline. Statistical analysis of peak amplitudes was performed using one-way repeated measures ANOVA (RM ANOVA) followed by Dunnett post hoc comparison tests.

Time–frequency (TF) analysis was also used to detect the energy of the oscillations in the brain evoked by the noxious electrical stimulus. TF analysis has been extensively used in functional studies of brain activity in humans and animal models.10,26,27  First, continuous intracortical activity was high-pass filtered at 0.5 Hz with a zero-phase second-order Butterworth filter. TF analysis was then performed using a complex Morse wavelet transform.26,28  This allowed us to calculate a complex TF spectral estimate W(a,b) of the intracortical activity at each point (a,b) of the TF plane from 3 s before the stimulus to 3 s after stimulus in the time domain and between 0.5 and 100 Hz (in logarithmic steps) in the frequency domain. The changes in TF spectral energy (i.e., modulus square) in the intracortical activity in response to the stimulation were estimated by normalizing to the mean energy content of the baseline period (a period of quiescence of 1 s) at each frequency.10  The TF energy, time locked to the stimulation in each group (anesthetic step, age, incision), was presented as a group median. The stimulus-induced energy changes, time locked to each stimulus, were estimated separately at each postnatal age and anesthetic step.

Results

Noxious-evoked Somatosensory Cortical Activity Is Resistant to Isoflurane Anesthesia in Neonatal Rats

We first recorded intracortical spontaneous activity in the primary somatosensory cortex of P7 rats after 40-min equilibration at 1.5% isoflurane and through subsequent step increases in inspired concentration (n = 7 animals). Figure 2A shows typical traces of S1 intracortical activity at each inspired isoflurane concentration. No spontaneous activity was recorded in any frequency band in the P7 primary somatosensory cortex at 1.5% inspired isoflurane or at higher concentrations (fig. 2B)

Fig. 2.

The effect of postnatal age and inspired isoflurane concentration on ongoing spontaneous somatosensory intracortical activity. (A) Typical 10-s traces of spontaneous activity at 1.5% inspired isoflurane (Iso 1.5%) and during step increases every 5 min in inspired isoflurane (step 1: 2%; step 2: 3%; step 3: 4%; step 4: 5%) in rats aged postnatal day (P) 7, P14, P21, and P30. (B) Spectral properties of intracortical activity (frequency range: 1 to 100 Hz) at each age and inspired isoflurane concentration. Spectral analysis (fast Fourier transformation, frequency range: 1 to 100 Hz) was performed on 100-s epochs. Mean ± SEM (n = 7 animals per age).

Fig. 2.

The effect of postnatal age and inspired isoflurane concentration on ongoing spontaneous somatosensory intracortical activity. (A) Typical 10-s traces of spontaneous activity at 1.5% inspired isoflurane (Iso 1.5%) and during step increases every 5 min in inspired isoflurane (step 1: 2%; step 2: 3%; step 3: 4%; step 4: 5%) in rats aged postnatal day (P) 7, P14, P21, and P30. (B) Spectral properties of intracortical activity (frequency range: 1 to 100 Hz) at each age and inspired isoflurane concentration. Spectral analysis (fast Fourier transformation, frequency range: 1 to 100 Hz) was performed on 100-s epochs. Mean ± SEM (n = 7 animals per age).

Despite the lack of spontaneous activity, hind paw electrical stimulation evoked clear low amplitude–evoked potentials, with simple positive–negative voltage waveforms, in the P7 somatosensory cortex (fig. 3). A significant decrease in the mean evoked potential amplitude did not occur until step 3, where it dropped to half the amplitude recorded at 1.5% inspired isoflurane (50 ± 9%; fig. 3, A and B). At the maximum inspired isoflurane concentration (step 4), evoked potentials were greatly diminished (6 ± 1%) but importantly were still clearly detectable in five of the seven animals studied. A TF analysis of this evoked activity is shown in figure 3C to demonstrate the mean energy across different frequencies (1 to 100 Hz) evoked in 3 s after noxious electrical stimulation. At 1.5% inspired isoflurane, a strong increase in energy at all frequencies is observed in the first 500 ms after stimulus, but this declines with increasing levels of inspired isoflurane, notably at steps 3 and 4 (fig. 3C). At step 4, the oscillation energy is greatly reduced, but still present.

Fig. 3.

Effect of inspired isoflurane concentration on noxious-evoked activity in the primary somatosensory cortex at postnatal day 7. (A) Typical recordings of right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line) during increasing inspired isoflurane (Iso) concentrations. Grand average evoked potentials (filled lines) ±SEM (gray area) from 10 stimuli per animal (n = 7 animals). (B) Bar chart showing the peak evoked potential amplitude at each Iso step, normalized to initial recordings during 1.5% Iso (repeated measures [RM] ANOVA F(4, 24) = 78, P < 0.0001; Dunnett post hoc test, ***P < 0.001, compared with activity during inspired 1.5% Iso). (C) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in A. The time–frequency energy changes, time locked to each stimulus, are presented as a group median. Results are displayed as increases and decreases in energy relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas values between 0 and 2 correspond to energy increases. n = 7 animals for each age group. (D) Evoked potentials as in A, but 10 min after plantar skin incision, (10 stimuli per animal in n = 6 animals). (E) Bar chart showing the peak evoked potential amplitude after hind paw incision at steps of increasing of inspired Iso concentration, normalized to recordings during 1.5% Iso (RM ANOVA F(4, 20) = 24, P < 0.0001; Dunnett post hoc test, **P < 0.01, compared with activity recorded during 1.5% Iso. (F) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in D, 10 min after plantar skin incision.

Fig. 3.

Effect of inspired isoflurane concentration on noxious-evoked activity in the primary somatosensory cortex at postnatal day 7. (A) Typical recordings of right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line) during increasing inspired isoflurane (Iso) concentrations. Grand average evoked potentials (filled lines) ±SEM (gray area) from 10 stimuli per animal (n = 7 animals). (B) Bar chart showing the peak evoked potential amplitude at each Iso step, normalized to initial recordings during 1.5% Iso (repeated measures [RM] ANOVA F(4, 24) = 78, P < 0.0001; Dunnett post hoc test, ***P < 0.001, compared with activity during inspired 1.5% Iso). (C) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in A. The time–frequency energy changes, time locked to each stimulus, are presented as a group median. Results are displayed as increases and decreases in energy relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas values between 0 and 2 correspond to energy increases. n = 7 animals for each age group. (D) Evoked potentials as in A, but 10 min after plantar skin incision, (10 stimuli per animal in n = 6 animals). (E) Bar chart showing the peak evoked potential amplitude after hind paw incision at steps of increasing of inspired Iso concentration, normalized to recordings during 1.5% Iso (RM ANOVA F(4, 20) = 24, P < 0.0001; Dunnett post hoc test, **P < 0.01, compared with activity recorded during 1.5% Iso. (F) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in D, 10 min after plantar skin incision.

We next tested whether hind paw incision influenced the sensitivity of plantar C-fiber–evoked potentials to step increases in anesthesia. The immediate effect of the plantar incision is shown in figure 4. In P7 rats, spontaneous activity was increased during, and immediately after, the incision (δ band activity, 0.02 ± 0.01 to 0.10 ± 0.02 μV/Hz; RM ANOVA F(3, 15) = 13.5, P = 0.0002; post hoc Dunnett test: P < 0.001, and β band activity (0.01 ± 0.00 to 0.02 ± 0.01 μV/Hz, RM ANOVA, F(3, 15) = 6.1, P = 0.0062; post hoc Dunnett test: P < 0.05), which returned to baseline at 10 min (fig. 4, A and B). In addition figure 3 shows that hind paw incision had a more persistent effect on the response to subsequent noxious electrical stimuli. In animals with previous hind paw incision, the mean peak amplitude of the electrical-evoked potential was not significantly altered by step increase in isoflurane level, except at step 4 (RM ANOVA F (4, 20) = 3.7, P = 0.0210; post hoc Dunnett test: P < 0.01 between baseline 1.5% and step 4), and even at the maximal inspired isoflurane concentration, the mean response remained at 67 ± 16% of the peak amplitude evoked at the initial 1.5% isoflurane concentration (fig. 3, D and E). This is further confirmed by the TF analysis (fig. 3F). At baseline 1.5% isoflurane and at step 1, the strong increase in energy at all frequencies in the first 500 ms after stimulus does not differ in animals with and without skin incision, but this is not so at higher levels of isoflurane. Thus, after hind paw incision, the energy of cortical activity does not diminish with increasing inspired concentration of isoflurane. The comparison of oscillation energies with and without plantar hind paw incision at step 4, shown in figure 3, shows that the presence of surgical injury increases the resistance of infant S1 cortex nociceptive activity to isoflurane (fig. 3, C and F, bottom panels).

Fig. 4.

The effect of skin incision on spontaneous activity in the somatosensory cortex in postnatal day (P) 7 and P30 rats. (A) Typical 10-s intracortical traces from P7 rats sampled from the 2-min interval before, during, and after plantar skin incision and again 10 min later. (B) Spectral analysis (fast Fourier transformation) from 100-s epochs quantified changes across the frequency range 1 to 100 Hz. The graph represents the amplitude (microvolts per Hertz) across the frequency range 1 to 100 Hz. Data are represented as mean ± SEM (n = 6 animals). A significant main effect of hind paw incision (before and during incision) was observed in the increased δ band activity (0.02 ± 0.01 to 0.10 ± 0.02 μV/Hz; repeated measures [RM] ANOVA F(3, 15) = 13, P = 0.0002; post hoc Dunnett test, P < 0.001) and β band activity (0.01 ± 0.00 to 0.02 ± 0.01 μV/Hz, RM ANOVA, F(3, 15) = 6, P = 0.0062; post hoc Dunnett test, P < 0.05). This had recovered by 10 min after surgery. (C) Typical 10-s intracortical traces from P30 sampled from the 2-min interval before, during, and after plantar skin incision and again 10 min later. (D) Spectral analysis in P30 rats. In contrast to P7 rats, the frequency distribution of the mean spontaneous activity at P30 (n = 6 animals at each age) in a 100-s epoch before, during, and after incision under 1.5% isoflurane anesthesia was unchanged (δ band from 1.05 ± 0.14 to 1.22 ± 0.12 μV/Hz, RM ANOVA F (3, 15) = 0.8, P = 0.4994, nonsignificant; β band from 0.01 ± 0.00 to 0.02 ± 0.01 μV/Hz, RM ANOVA F (3, 15) = 0.7, P = 0.5688, nonsignificant).

Fig. 4.

The effect of skin incision on spontaneous activity in the somatosensory cortex in postnatal day (P) 7 and P30 rats. (A) Typical 10-s intracortical traces from P7 rats sampled from the 2-min interval before, during, and after plantar skin incision and again 10 min later. (B) Spectral analysis (fast Fourier transformation) from 100-s epochs quantified changes across the frequency range 1 to 100 Hz. The graph represents the amplitude (microvolts per Hertz) across the frequency range 1 to 100 Hz. Data are represented as mean ± SEM (n = 6 animals). A significant main effect of hind paw incision (before and during incision) was observed in the increased δ band activity (0.02 ± 0.01 to 0.10 ± 0.02 μV/Hz; repeated measures [RM] ANOVA F(3, 15) = 13, P = 0.0002; post hoc Dunnett test, P < 0.001) and β band activity (0.01 ± 0.00 to 0.02 ± 0.01 μV/Hz, RM ANOVA, F(3, 15) = 6, P = 0.0062; post hoc Dunnett test, P < 0.05). This had recovered by 10 min after surgery. (C) Typical 10-s intracortical traces from P30 sampled from the 2-min interval before, during, and after plantar skin incision and again 10 min later. (D) Spectral analysis in P30 rats. In contrast to P7 rats, the frequency distribution of the mean spontaneous activity at P30 (n = 6 animals at each age) in a 100-s epoch before, during, and after incision under 1.5% isoflurane anesthesia was unchanged (δ band from 1.05 ± 0.14 to 1.22 ± 0.12 μV/Hz, RM ANOVA F (3, 15) = 0.8, P = 0.4994, nonsignificant; β band from 0.01 ± 0.00 to 0.02 ± 0.01 μV/Hz, RM ANOVA F (3, 15) = 0.7, P = 0.5688, nonsignificant).

To test whether this effect of skin incision was nociceptive specific, we compared the effects on potentials evoked by low-intensity innocuous A-fiber stimulation versus those evoked by high-intensity noxious C-fiber stimulation in the same animal. Figure 5, A and B show that, while in intact skin A-fiber stimulation evokes distinct potentials that slowly diminish with the increasing levels of isoflurane, after skin incision, the A-fiber–evoked response is completely absent at all isoflurane levels (fig. 5, C and D). This is in marked contrast to the C-fiber–evoked potentials that persist unchanged after skin incision at all but the highest step level of isoflurane (fig. 5, E and F).

Fig. 5.

Effect of isoflurane (Iso) and hind paw incision on nonnoxious, A-fiber (0.32 mA, 50 μs) and noxious, C-fiber (3.2 mA, 500 μs) evoked potentials in the primary somatosensory cortex at postnatal day (P) 7. (A) Typical recordings of right somatosensory-evoked potentials (SEPs) after low-intensity A-fiber electrical stimulation of the left hind paw (10 × 0.32 mA, 50 μs stimuli) applied at the time indicated by the dotted line during increasing inspired Iso concentrations. Grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 5 animals). (B) Bar chart showing the peak evoked potential amplitude at each Iso step, normalized to initial recordings during 1.5% Iso (Pre Iso 1.5%; repeated measures [RM] ANOVA F(5, 20) = 40, P < 0.0001; Dunnett post hoc test, *P < 0.05; ***P < 0.001, compared with activity during Pre Iso 1.5%. C and E show typical recordings of P7 right SEPs in response to (C) low-intensity, A-fiber (C, 5 × 0.32 mA, 50 μs stimuli) and (E) high-intensity, C-fiber (5 × 3.2 mA, 500 μs) electrical stimulation of the left hind paw after hind paw incision at increasing inspired Iso steps. Traces represent grand average evoked potentials (filled lines) ± SEM (gray area) from 5 stimuli (5 × low intensity, and 5 × high intensity) per animal (n = 5 animals). The dotted line indicates the time of electrical stimulation. D and F show bar charts of the peak evoked potential amplitude at each Iso step with low-intensity electrical stimulation (D) and high-intensity electrical stimulation (F). The data were normalized to initial recordings during 1.5% Iso (Pre Iso 1.5%; for low-intensity stimulation: RM ANOVA F(5, 20) = 1673, P < 0.0001; for high-intensity stimulation: RM ANOVA F(5, 20) = 6, P = 0.0011, Dunnett post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001, compared to activity during Pre Iso 1.5%).

Fig. 5.

Effect of isoflurane (Iso) and hind paw incision on nonnoxious, A-fiber (0.32 mA, 50 μs) and noxious, C-fiber (3.2 mA, 500 μs) evoked potentials in the primary somatosensory cortex at postnatal day (P) 7. (A) Typical recordings of right somatosensory-evoked potentials (SEPs) after low-intensity A-fiber electrical stimulation of the left hind paw (10 × 0.32 mA, 50 μs stimuli) applied at the time indicated by the dotted line during increasing inspired Iso concentrations. Grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 5 animals). (B) Bar chart showing the peak evoked potential amplitude at each Iso step, normalized to initial recordings during 1.5% Iso (Pre Iso 1.5%; repeated measures [RM] ANOVA F(5, 20) = 40, P < 0.0001; Dunnett post hoc test, *P < 0.05; ***P < 0.001, compared with activity during Pre Iso 1.5%. C and E show typical recordings of P7 right SEPs in response to (C) low-intensity, A-fiber (C, 5 × 0.32 mA, 50 μs stimuli) and (E) high-intensity, C-fiber (5 × 3.2 mA, 500 μs) electrical stimulation of the left hind paw after hind paw incision at increasing inspired Iso steps. Traces represent grand average evoked potentials (filled lines) ± SEM (gray area) from 5 stimuli (5 × low intensity, and 5 × high intensity) per animal (n = 5 animals). The dotted line indicates the time of electrical stimulation. D and F show bar charts of the peak evoked potential amplitude at each Iso step with low-intensity electrical stimulation (D) and high-intensity electrical stimulation (F). The data were normalized to initial recordings during 1.5% Iso (Pre Iso 1.5%; for low-intensity stimulation: RM ANOVA F(5, 20) = 1673, P < 0.0001; for high-intensity stimulation: RM ANOVA F(5, 20) = 6, P = 0.0011, Dunnett post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001, compared to activity during Pre Iso 1.5%).

Effects of Isoflurane on Spontaneous Intracortical Activity Are Influenced by Postnatal Age

We next examined the effect of postnatal age on the relationship between primary somatosensory cortical spontaneous and evoked activity at increasing inspired isoflurane concentrations. To do this, the same experiment was performed at P14, P21, and P30, and the results were compared with those obtained at P7.

Figure 2 shows the intracortical spontaneous activity in the primary somatosensory cortex of the rat, at the four postnatal ages after equilibration at 1.5% isoflurane and through subsequent step increases in inspired isoflurane concentration (n = 7 animals at each age). In contrast to the lack of spontaneous activity at P7, figure 2A shows that at P14, P21, and P30, an inspired isoflurane concentration of 1.5% induced burst suppression activity in the somatosensory cortex, characterized by intermittent, highly synchronized neuronal discharges (bursts) separated by silent periods (suppression), as described elsewhere.29  The burst suppression activity disappears as inspired isoflurane concentration increases. Figure 2B shows the distribution of frequency components of spontaneous somatosensory cortical activity at each age. At P14 to P30, activity across all frequencies decreases steadily as the inspired isoflurane concentration increases, and there is no spontaneous activity and an isoelectric intracortical activity by step 4.

Noxious-evoked Activity in the Somatosensory Cortex Is More Sensitive to Increasing Isoflurane Concentration in Older Rats

We next compared the primary somatosensory (S1) cortical potentials evoked by noxious, C-fiber intensity electrical stimuli in the contralateral hind paw (10 stimuli/step/animal, n = 7 animals) in P7, P14, P21, and P30 rats during increasing inspired isoflurane concentrations (fig. 6). Sample traces are shown in figure 6A, and the mean peak amplitude of the evoked potential is plotted in figure 6B, as the percentage decrease with each step increase in isoflurane, normalized to the initial values recorded after equilibration at 1.5% isoflurane. The relative decrease in evoked potential peak amplitude with each step increase in isoflurane is more marked in older animals (P14, P21, and P30) than at P7. At step 3, the mean peak amplitude has reduced to half in P7 animals (50 ± 9% of initial 1.5% isoflurane values), in contrast to P21 and P30 where it drops below 10% (P21: 6 ± 1%; P30: 4 ± 1%). At Step 4, no discernible evoked potentials were recorded from any P21 or P30 animals but were recorded from three of seven P14 animals and five of seven P7 animals.

Fig. 6.

The effect of postnatal age on somatosensory-evoked potentials evoked by C-fiber electrical hind paw skin stimulation. (A) Typical recordings of right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line). Recordings were performed at 1.5% inspired isoflurane (Iso 1.5%) and during 5-min step increases (step 1: 2%; step 2: 3%; step 3: 4%; step 4: 5%) in rats aged postnatal day (P) 7, P14, P21, and P30. Traces represent grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 7 animals). Note the different y-axis scale at P7 compared with older ages. (B) Comparison of the evoked potential peak amplitude at each Iso step, normalized to initial values obtained during Iso 1.5% (repeated measures ANOVA, P7: F(4, 24) = 78, P < 0.0001; P14: F(4, 24) = 37, P < 0.0001; P21: F(4, 24) = 17, P = 0.0008; P30: F(4, 24) =49, P < 0.0001 and Dunnett post hoc multiple comparisons test: **P < 0.01, and ***P < 0.001, compared with evoke peak amplitude during inspired Iso 1.5%).

Fig. 6.

The effect of postnatal age on somatosensory-evoked potentials evoked by C-fiber electrical hind paw skin stimulation. (A) Typical recordings of right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line). Recordings were performed at 1.5% inspired isoflurane (Iso 1.5%) and during 5-min step increases (step 1: 2%; step 2: 3%; step 3: 4%; step 4: 5%) in rats aged postnatal day (P) 7, P14, P21, and P30. Traces represent grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 7 animals). Note the different y-axis scale at P7 compared with older ages. (B) Comparison of the evoked potential peak amplitude at each Iso step, normalized to initial values obtained during Iso 1.5% (repeated measures ANOVA, P7: F(4, 24) = 78, P < 0.0001; P14: F(4, 24) = 37, P < 0.0001; P21: F(4, 24) = 17, P = 0.0008; P30: F(4, 24) =49, P < 0.0001 and Dunnett post hoc multiple comparisons test: **P < 0.01, and ***P < 0.001, compared with evoke peak amplitude during inspired Iso 1.5%).

Figure 7 shows TF analysis of evoked activity at each age. The mean energy of cortical activity across different frequencies (1 to 100 Hz) evoked in the 3 s after noxious electrical stimulation is shown at the four steps of increasing inspired isoflurane (10 stimuli/step/animal, n = 7 animals for each age group). At all ages, the evoked response energy decreases with increasing inspired isoflurane. Although noxious-evoked cortical activity is relatively resistant to increasing isoflurane concentration at P7, there is a gradual increase in the degree of suppression by isoflurane at older ages (fig. 7).

Fig. 7.

The effect of postnatal age on cortical oscillatory activity after C-fiber electrical hind paw skin stimulation. Time– frequency decomposition of the evoked somatosensory cortical neural activity at each isoflurane step at postnatal day (P) 7, P14, P21, and P30. The time–frequency energy changes, time locked to each C-fiber stimulus, are presented as a group median (10 stimuli per animal, n = 7 animals). Results are displayed as increases and decreases of energy changes relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas the values between 0 and 2 correspond to energy increases (n = 7 animals for each age group).

Fig. 7.

The effect of postnatal age on cortical oscillatory activity after C-fiber electrical hind paw skin stimulation. Time– frequency decomposition of the evoked somatosensory cortical neural activity at each isoflurane step at postnatal day (P) 7, P14, P21, and P30. The time–frequency energy changes, time locked to each C-fiber stimulus, are presented as a group median (10 stimuli per animal, n = 7 animals). Results are displayed as increases and decreases of energy changes relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas the values between 0 and 2 correspond to energy increases (n = 7 animals for each age group).

The Effect of Hind Paw Incision on Cortical Nociceptive Activity at P30 Differs from that at P7

As surgical incision had a profound effect on the subsequent cortical response to noxious electrical stimulation at P7, we tested whether this also occurred at an older age. Figure 4 shows that there was no immediate effect of the plantar incision on spontaneous activity during, and immediately after, the incision at P30 (fig. 4, C and D). Figure 8 illustrates the effect of plantar incision on nociceptive-evoked potential at each anesthetic step in P30 rats. Electrical-evoked nociceptive potentials are progressively suppressed by increasing inspired isoflurane concentration in the absence (fig. 8, A and B) and presence (fig. 8, C and D) of skin incision. The reduction in peak evoked potential amplitude produced by step increases in inspired isoflurane (fig. 8B) was not altered after hind paw incision (fig. 8D). There was a steady fall in mean evoked potential amplitude, expressed as a percentage change from baseline (1.5% isoflurane) in both nonincised (step 1, 88 ± 14; step 2, 62 ± 8; step 3, 4 ± 1; step 4, 1.4 ± 0.5, F (4, 20) = 49, P < 0.0001) and incised groups (step 1, 62 ± 9; step 2, 50 ± 19; step 3, 3.5 ± 1; step 4, 0.13 ± 0.1, F (4, 20) = 25, P < 0.0001) (fig. 8, B and D). TF analysis (fig. 8, E and F) shows that skin incision at 1.5% isoflurane causes an increased energy and duration of evoked cortical oscillations at P30, not seen at P7, suggesting some underlying sensitization of cortical pain circuits. Although clear at an inspired isoflurane concentration of 1.5%, this increased cortical response is highly sensitive to the subsequent increases in isoflurane, and the difference in response between P30 animals with (fig. 8F) and without (fig. 8E) hind paw incision is largely lost at step 1, and evoked responses in both groups are significantly diminished at steps 2 and 3 and effectively gone at step 4. These results differ significantly from those obtained at P7 (fig. 3), where cortical-evoked potentials and oscillatory activity persisted and were resistant to increasing inspired isoflurane concentration.

Fig. 8.

Effect of isoflurane and hind paw incision on noxious-evoked activity in primary somatosensory cortex at postnatal day (P) 30. (A) Typical recordings of P30 right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line) during increasing inspired isoflurane (Iso) concentrations. Grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 7 animals). (B) Bar chart showing the peak evoked potential amplitude at each isoflurane step, normalized to initial recordings during 1.5% Iso (repeated measures [RM] ANOVA F(4, 24) = 49, P < 0.0001; Dunnett post hoc test, **P < 0.01, and ***P < 0.001, compared with peak evoked potential amplitude during 1.5% Iso). (C) P30 evoked potentials as in A, but 10 min after plantar skin incision (10 stimuli per animal in n = 6 animals). (D) Bar chart showing the peak evoked potential amplitude after hind paw incision at steps of increasing of inspired isoflurane concentration, normalized to recordings during 1.5% Iso (RM ANOVA F(4, 20) = 24, P < 0.0001; Dunnett post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, compared with activity recorded during 1.5% Iso. (E) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in A. The time–frequency energy changes, time locked to each stimulus, are presented as a group median. Results are displayed as increases and decreases in energy relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas values between 0 and 2 correspond to energy increases. n = 7 animals for each age group. (F) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in C, 10 min after plantar skin incision.

Fig. 8.

Effect of isoflurane and hind paw incision on noxious-evoked activity in primary somatosensory cortex at postnatal day (P) 30. (A) Typical recordings of P30 right somatosensory-evoked potentials after electrical stimulation of the left hind paw (10 × 3.2 mA, 500 μs stimuli applied at the time indicated by the dotted line) during increasing inspired isoflurane (Iso) concentrations. Grand average evoked potentials (filled lines) ± SEM (gray area) from 10 stimuli per animal (n = 7 animals). (B) Bar chart showing the peak evoked potential amplitude at each isoflurane step, normalized to initial recordings during 1.5% Iso (repeated measures [RM] ANOVA F(4, 24) = 49, P < 0.0001; Dunnett post hoc test, **P < 0.01, and ***P < 0.001, compared with peak evoked potential amplitude during 1.5% Iso). (C) P30 evoked potentials as in A, but 10 min after plantar skin incision (10 stimuli per animal in n = 6 animals). (D) Bar chart showing the peak evoked potential amplitude after hind paw incision at steps of increasing of inspired isoflurane concentration, normalized to recordings during 1.5% Iso (RM ANOVA F(4, 20) = 24, P < 0.0001; Dunnett post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, compared with activity recorded during 1.5% Iso. (E) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in A. The time–frequency energy changes, time locked to each stimulus, are presented as a group median. Results are displayed as increases and decreases in energy relative to a baseline period of 1 s before stimulation. Energy values between 0 and −2 correspond to energy decreases, whereas values between 0 and 2 correspond to energy increases. n = 7 animals for each age group. (F) Time–frequency decomposition of the evoked somatosensory cortical neural activity shown in C, 10 min after plantar skin incision.

Discussion

The primary aim of this study was to test the impact of increasing isoflurane concentration on spontaneous and evoked nociceptive activity in the somatosensory cortex of infant rats undergoing hind paw incision. We hypothesized that, in infant rats, noxious-evoked brain activity is more resistant than spontaneous brain activity to isoflurane anesthesia. The results, obtained by recording intracortical neuronal activity from layer 5 to 6 of the somatosensory cortex in P7 rats during increasing inspired concentrations of isoflurane, support this hypothesis. The data show that isoflurane influences spontaneous activity and evoked activity in the infant rat somatosensory cortex quite differently.

At P7, all concentrations of inspired isoflurane (1.5 to 5%) silenced cortical neurons and suppressed spontaneous bursts and oscillations. This effect is consistent with a previous study in the neonatal somatosensory cortex, where cortical activity was completely suppressed in P7 animals by 1.5 to 2% isoflurane.16  This is in contrast to the spontaneous cortical activity observed in awake or very lightly anesthetized animals at this age, which is characterized by intermittent bursts characterized by α–β (spindle bursts) and γ frequency bands (early γ oscillations).16,30–33 

In contrast to the absence of spontaneous activity, noxious somatosensory–evoked potentials and evoked oscillatory activity persisted, even at high inspired concentrations of isoflurane. This result highlights the differences in the neural mechanisms generating spontaneous activity and evoked activity in the immature somatosensory cortex. Activity in the developing brain changes with age, and early cortical development is marked by unique patterns of activity as functional circuits mature.34,35  Early oscillatory bursts are generated in thalamocortical circuits32,36  but can also be triggered by sensory inputs and are likely to be generated by glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-d-aspartate receptors, whereas GABAergic (γ-aminobutyric acid releasing) interneurons compartmentalize the activated areas via surround inhibition.37,38  The transition from predominantly nonspecific neuronal bursts to specific cortical sensory evoked potentials seems to be triggered by increasing sensory input31  in the neonatal rodent somatosensory cortex.16  A similar transition begins in the preterm human infant brain at around 35 weeks postconceptional age.26,39  Thus, the nociceptive-evoked activity in the newborn infant cortex recorded at P7 is still relatively immature and may be less tightly coupled within specific networks, rendering it less susceptible to suppression by volatile anesthetics than at older ages.

A further finding here is that the relative insensitivity of noxious-evoked activity to isoflurane was enhanced after plantar hind paw incision. This well-established model of surgical incision pain,21  adapted for younger rat pups,40,41  rendered infant rat nociceptive-evoked cortical potentials and oscillatory network activity totally resistant to the maximum inspired isoflurane tested (5%). The mechanism for this is not clear but may reflect widespread depolarization of central neurons by the incoming nociceptive barrage. It is also notable that the incision itself increased cortical activity in young animals, consistent with the reported spike activity and sensitization of receptive fields in infant dorsal horn cells after hind paw incision.42  Importantly, the enhanced isoflurane resistance after skin incision at P7 was selective for C-fiber nociceptive-evoked potentials. In contrast, A-fiber innocuous evoked potentials were completely abolished after skin incision at all isoflurane levels, reflecting the different sensitivities of immature A- and C-fiber–evoked activity to skin incision.43 

A secondary outcome of this study was the significant age dependence of the isoflurane effects on spontaneous and noxious-evoked activity in the somatosensory cortex. At P14 and older ages, 1.5% isoflurane produced a typical pattern of burst suppression with high-amplitude low-frequency activity and a progressive decrease in spontaneous activity with step increases in inspired isoflurane concentration, as reported elsewhere.29,44,45  This contrasted with the suppression of spontaneous spindle bursts and γ oscillations even at the initial inspired concentration of 1.5% isoflurane at P7. More important was the greater sensitivity of the noxious evoked potentials to isoflurane in older animals. As reported in adult rats9  noxious-evoked potentials at P14 to P30 decreased markedly with increasing concentrations of inspired isoflurane and at P21 and P30 were completely absent at 5% isoflurane. Differences were especially clear using TF analysis, which reveals the energy and power of oscillating signals in the cortex, and thus the changes within different frequency bands that are linked to specific sensory and motor functions.25  This form of analysis has been used during isoflurane anesthesia in adult rats to characterize concentration-dependent changes in both low (30 to 50 Hz) and high (70 to 140 Hz) frequency γ power in different brain regions.44 

A further secondary outcome was the failure of surgical incision to affect noxious-evoked potential sensitivity to isoflurane at the older age of P30. Unlike the P7 rat, surgical incision itself at P30 caused no immediate cortical activity but did have a sensitizing effect on noxious cortical–evoked activity at 1.5% isoflurane. This is consistent with previous reports of increased C-fiber input to the adult rat somatosensory cortex in a UV-B irradiation model of hyperalgesia.8  However, in contrast to P7 rats, the reduction of evoked potentials and oscillatory activity by isoflurane were not altered by surgical incision in rats at P30. These data suggest that the powerful effect at P7 is not due to the effects of central sensitization, but a different yet unknown effect.

Our aim of this study was not to compare “hypnotic potency” at the different ages, but rather to evaluate within age group changes in cortical activity during isoflurane anesthesia. Equipotent concentrations of volatile anesthetics have been traditionally based on the minimum alveolar concentration (MAC) that prevents movement to a standardized noxious stimuli. However, these values do not reflect hypnotic potency in the brain.46  The MAC of isoflurane is higher in P7 to P9 rodents,47,48  as spinal reflex excitability is greater at this age, but a clear stimulus–response relationship that is sensitive to injury, analgesia, and inspired volatile agent concentration is evident across all ages.22,49  Although isoflurane actions in the spinal cord may reduce ascending somatosensory information and indirectly alter cortical activity,50  the level of cortical-evoked activity cannot be inferred from the presence or absence of a visible reflex response. Here, hind paw incision (but not electrical stimulation) produced a brief visible reflex response in P7 but not older animals, consistent with the reported greater MAC, but in older animals noxious stimuli produced clear cortical-evoked responses at 1.5% isoflurane, despite a lack of reflex response. The age dependence of cortical-evoked response sensitivity to isoflurane may have been influenced by alterations in physiologic parameters. Heart rate was monitored throughout the experiments and even in the more prolonged protocols including hind paw incision; decreases were only seen at the highest level of inspired isoflurane and to the same degree (approximately 20% reduction) in P7 and P30 groups. Animals were mechanically ventilated in isoflurane and oxygen with age-adjusted settings, and although we cannot confirm that partial pressures of carbon dioxide (Pco2) were the same across the different ages, changes with increasing isoflurane concentration would not have been influenced by respiratory parameters.

These rat data obtained have considerable implications for clinical practice. The timing and sequence of key events in brain development are exceptionally similar across mammals, and a recent neuroinformatic analysis shows that sensorimotor events in the cortex of the P7 rat translate to 1 to 2 months in the human.51  This study clearly demonstrates that spontaneous intracortical network activity is more effectively silenced by isoflurane in the neonatal brain, but that isoflurane has less effect on cortical activity evoked by peripheral noxious sensory inputs. As the developing central nervous system is vulnerable to changes in neural activity, maintaining appropriate anesthesia in infants is likely to require avoidance of both excessive reductions in cortical activity that may enhance neuronal apoptosis and excessive increases in activity due to uncontrolled noxious inputs.1–4  Anesthesia-induced developmental neurotoxicity has been clearly demonstrated in neonatal animals,3  and the associated long-lasting cognitive impairment has raised concern for young children undergoing anesthesia.4  The data here highlight the complexity of using intracortical activity parameters to define or measure the level of anesthesia required to produce “hypnosis” in neonates and infants. The persistence of noxious sensory–evoked responses despite increasing isoflurane concentration in young rats emphasizes the critical need to provide analgesia in neonates and infants. Surgery and tissue injury in neonatal rodents can produce long-term changes in sensory processing,52  but this can be modified by morphine53,54  or local anesthetic blockade.40,55  Increased understanding of age- and anesthesia-dependent changes in intracortical activity may improve algorithms for evaluating depth of anesthesia in neonates and infants, and comparative studies of the ability of different types and doses of analgesic to minimize noxious-evoked responses are likely to improve acute clinical care and long-term neurodevelopmental outcome after neonatal surgery.

Acknowledgments

The authors thank Tom Carson, B.Sc., Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom, for his technical support.

This study was supported by the Medical Research Council, London, United Kingdom, grants G0901269 (to Dr. Fitzgerald) and MR/K022636/1 (to Dr. Walker).

Competing Interests

The authors declare no competing interests.

References

1.
Sanders
RD
,
Hassell
J
,
Davidson
AJ
,
Robertson
NJ
,
Ma
D
:
Impact of anaesthetics and surgery on neurodevelopment: An update.
Br J Anaesth
2013
;
110
(
suppl 1
):
i53
72
2.
Lin
EP
,
Soriano
SG
,
Loepke
AW
:
Anesthetic neurotoxicity.
Anesthesiol Clin
2014
;
32
:
133
55
3.
Liu
J
,
Rossaint
R
,
Sanders
RD
,
Coburn
M
:
Toxic and protective effects of inhaled anaesthetics on the developing animal brain: Systematic review and update of recent experimental work.
Eur J Anaesthesiol
2014
;
31
:
669
77
4.
Warner
DO
,
Flick
RP
:
Effects of anesthesia and surgery on the developing brain: Problem solved?
Paediatr Anaesth
2015
;
25
:
435
6
5.
Brummelte
S
,
Grunau
RE
,
Chau
V
,
Poskitt
KJ
,
Brant
R
,
Vinall
J
,
Gover
A
,
Synnes
AR
,
Miller
SP
:
Procedural pain and brain development in premature newborns.
Ann Neurol
2012
;
71
:
385
96
6.
Walker
SM
:
Biological and neurodevelopmental implications of neonatal pain.
Clin Perinatol
2013
;
40
:
471
91
7.
Schwaller
F
,
Fitzgerald
M
:
The consequences of pain in early life: Injury-induced plasticity in developing pain pathways.
Eur J Neurosci
2014
;
39
:
344
52
8.
Jensen
T
,
Granmo
M
,
Schouenborg
J
:
Altered nociceptive C fibre input to primary somatosensory cortex in an animal model of hyperalgesia.
Eur J Pain
2011
;
15
:
368
75
9.
Granmo
M
,
Jensen
T
,
Schouenborg
J
:
Nociceptive transmission to rat primary somatosensory cortex—Comparison of sedative and analgesic effects.
PLoS One
2013
;
8
:
e53966
10.
Fabrizi
L
,
Williams
G
,
Lee
A
,
Meek
J
,
Slater
R
,
Olhede
S
,
Fitzgerald
M
:
Cortical activity evoked by an acute painful tissue-damaging stimulus in healthy adult volunteers.
J Neurophysiol
2013
;
109
:
2393
403
11.
Iannetti
GD
,
Baumgärtner
U
,
Tracey
I
,
Treede
RD
,
Magerl
W
:
Pinprick-evoked brain potentials: A novel tool to assess central sensitization of nociceptive pathways in humans.
J Neurophysiol
2013
;
110
:
1107
16
12.
Baumgärtner
U
,
Greffrath
W
,
Treede
RD
:
Contact heat and cold, mechanical, electrical and chemical stimuli to elicit small fiber-evoked potentials: Merits and limitations for basic science and clinical use.
Neurophysiol Clin
2012
;
42
:
267
80
13.
Slater
R
,
Worley
A
,
Fabrizi
L
,
Roberts
S
,
Meek
J
,
Boyd
S
,
Fitzgerald
M
:
Evoked potentials generated by noxious stimulation in the human infant brain.
Eur J Pain
2010
;
14
:
321
6
14.
Verriotis
M
,
Fabrizi
L
,
Lee
A
,
Ledwidge
S
,
Meek
J
,
Fitzgerald
M
:
Cortical activity evoked by inoculation needle prick in infants up to one-year old.
Pain
2015
;
156
:
222
30
15.
Roth
D
,
Petersen-Felix
S
,
Bak
P
,
Arendt-Nielsen
L
,
Fischer
M
,
Bjerring
P
,
Zbinden
AM
:
Analgesic effect in humans of subanaesthetic isoflurane concentrations evaluated by evoked potentials.
Br J Anaesth
1996
;
76
:
38
42
16.
Sitdikova
G
,
Zakharov
A
,
Janackova
S
,
Gerasimova
E
,
Lebedeva
J
,
Inacio
AR
,
Zaynutdinova
D
,
Minlebaev
M
,
Holmes
GL
,
Khazipov
R
:
Isoflurane suppresses early cortical activity.
Ann Clin Transl Neurol
2014
;
1
:
15
26
17.
de Prost
N
,
Ricard
J-D
,
Saumon
G
,
Dreyfuss
D
:
Ventilator-induced lung injury: Historical perspectives and clinical implications.
Ann Intensive Care
2011
;
1
:
28
18.
Paxinos
G
,
Watson
C
:
The Rat Brain in Stereotaxic Coordinates
, 7th edition.
San Diego
,
Academic Press
,
2013
19.
Paxinos
G
,
Ashwell
KW
,
Tork
I
:
Atlas of the Developing Rat Nervous System
.
London
,
Academic Press
,
1994
20.
Jennings
E
,
Fitzgerald
M
:
Postnatal changes in responses of rat dorsal horn cells to afferent stimulation: A fibre-induced sensitization.
J Physiol
1998
;
509
(
pt 3
):
859
68
21.
Brennan
TJ
,
Vandermeulen
EP
,
Gebhart
GF
:
Characterization of a rat model of incisional pain.
Pain
1996
;
64
:
493
501
22.
Walker
SM
,
Tochiki
KK
,
Fitzgerald
M
:
Hindpaw incision in early life increases the hyperalgesic response to repeat surgical injury: Critical period and dependence on initial afferent activity.
Pain
2009
;
147
:
99
106
24.
Rampil
IJ
:
A primer for EEG signal processing in anesthesia.
Anesthesiology
1998
;
89
:
980
1002
25.
Akam
T
,
Kullmann
DM
:
Oscillatory multiplexing of population codes for selective communication in the mammalian brain.
Nat Rev Neurosci
2014
;
15
:
111
22
26.
Fabrizi
L
,
Slater
R
,
Worley
A
,
Meek
J
,
Boyd
S
,
Olhede
S
,
Fitzgerald
M
:
A shift in sensory processing that enables the developing human brain to discriminate touch from pain.
Curr Biol
2011
;
21
:
1552
8
27.
Narayanan
NS
,
Cavanagh
JF
,
Frank
MJ
,
Laubach
M
:
Common medial frontal mechanisms of adaptive control in humans and rodents.
Nat Neurosci
2013
;
16
:
1888
95
28.
Olhede
SC
,
Walden
AT
:
Generalized morse wavelets.
IEEE Trans Signal Process
2002
;
50
:
2661
70
29.
Ferron
JF
,
Kroeger
D
,
Chever
O
,
Amzica
F
:
Cortical inhibition during burst suppression induced with isoflurane anesthesia.
J Neurosci
2009
;
29
:
9850
60
30.
Khazipov
R
,
Sirota
A
,
Leinekugel
X
,
Holmes
GL
,
Ben-Ari
Y
,
Buzsáki
G
:
Early motor activity drives spindle bursts in the developing somatosensory cortex.
Nature
2004
;
432
:
758
61
31.
Colonnese
MT
,
Kaminska
A
,
Minlebaev
M
,
Milh
M
,
Bloem
B
,
Lescure
S
,
Moriette
G
,
Chiron
C
,
Ben-Ari
Y
,
Khazipov
R
:
A conserved switch in sensory processing prepares developing neocortex for vision.
Neuron
2010
;
67
:
480
98
32.
Yang
JW
,
An
S
,
Sun
JJ
,
Reyes-Puerta
V
,
Kindler
J
,
Berger
T
,
Kilb
W
,
Luhmann
HJ
:
Thalamic network oscillations synchronize ontogenetic columns in the newborn rat barrel cortex.
Cereb Cortex
2013
;
23
:
1299
316
33.
Tiriac
A
,
Uitermarkt
BD
,
Fanning
AS
,
Sokoloff
G
,
Blumberg
MS
:
Rapid whisker movements in sleeping newborn rats.
Curr Biol
2012
;
22
:
2075
80
34.
Blankenship
AG
,
Feller
MB
:
Mechanisms underlying spontaneous patterned activity in developing neural circuits.
Nat Rev Neurosci
2010
;
11
:
18
29
35.
Khazipov
R
,
Luhmann
HJ
:
Early patterns of electrical activity in the developing cerebral cortex of humans and rodents.
Trends Neurosci
2006
;
29
:
414
8
36.
Minlebaev
M
,
Colonnese
M
,
Tsintsadze
T
,
Sirota
A
,
Khazipov
R
:
Early γ oscillations synchronize developing thalamus and cortex.
Science
2011
;
334
:
226
9
37.
Minlebaev
M
,
Ben-Ari
Y
,
Khazipov
R
:
Network mechanisms of spindle-burst oscillations in the neonatal rat barrel cortex in vivo.
J Neurophysiol
2007
;
97
:
692
700
38.
Minlebaev
M
,
Ben-Ari
Y
,
Khazipov
R
:
NMDA receptors pattern early activity in the developing barrel cortex in vivo.
Cereb Cortex
2009
;
19
:
688
96
39.
Hrbek
A
,
Karlberg
P
,
Olsson
T
:
Development of visual and somatosensory evoked responses in pre-term newborn infants.
Electroencephalogr Clin Neurophysiol
1973
;
34
:
225
32
40.
Walker
SM
,
Tochiki
KK
,
Fitzgerald
M
:
Hindpaw incision in early life increases the hyperalgesic response to repeat surgical injury: Critical period and dependence on initial afferent activity.
Pain
2009
;
147
:
99
106
41.
Beggs
S
,
Currie
G
,
Salter
MW
,
Fitzgerald
M
,
Walker
SM
:
Priming of adult pain responses by neonatal pain experience: Maintenance by central neuroimmune activity.
Brain
2012
;
135
(
pt 2
):
404
17
42.
Ririe
DG
,
Bremner
LR
,
Fitzgerald
M
:
Comparison of the immediate effects of surgical incision on dorsal horn neuronal receptive field size and responses during postnatal development.
Anesthesiology
2008
;
109
:
698
706
43.
Boada
MD
,
Gutierrez
S
,
Giffear
K
,
Eisenach
JC
,
Ririe
DG
:
Skin incision-induced receptive field responses of mechanosensitive peripheral neurons are developmentally regulated in the rat.
J Neurophysiol
2012
;
108
:
1122
9
44.
Hudetz
AG
,
Vizuete
JA
,
Pillay
S
:
Differential effects of isoflurane on high-frequency and low-frequency γ oscillations in the cerebral cortex and hippocampus in freely moving rats.
Anesthesiology
2011
;
114
:
588
95
45.
Kortelainen
J
,
Jia
X
,
Seppänen
T
,
Thakor
N
:
Increased electroencephalographic gamma activity reveals awakening from isoflurane anaesthesia in rats.
Br J Anaesth
2012
;
109
:
782
9
46.
Palanca
BJ
,
Mashour
GA
,
Avidan
MS
:
Processed electroencephalogram in depth of anesthesia monitoring.
Curr Opin Anaesthesiol
2009
;
22
:
553
9
47.
Orliaguet
G
,
Vivien
B
,
Langeron
O
,
Bouhemad
B
,
Coriat
P
,
Riou
B
:
Minimum alveolar concentration of volatile anesthetics in rats during postnatal maturation.
Anesthesiology
2001
;
95
:
734
9
48.
Stratmann
G
,
Sall
JW
,
Eger
EI
II
,
Laster
MJ
,
Bell
JS
,
May
LD
,
Eilers
H
,
Krause
M
,
Heusen
Fv
,
Gonzalez
HE
:
Increasing the duration of isoflurane anesthesia decreases the minimum alveolar anesthetic concentration in 7-day-old but not in 60-day-old rats.
Anesth Analg
2009
;
109
:
801
6
49.
Walker
SM
,
Fitzgerald
M
:
Characterization of spinal alpha-adrenergic modulation of nociceptive transmission and hyperalgesia throughout postnatal development in rats.
Br J Pharmacol
2007
;
151
:
1334
42
50.
Antognini
JF
,
Jinks
SL
,
Atherley
R
,
Clayton
C
,
Carstens
E
:
Spinal anaesthesia indirectly depresses cortical activity associated with electrical stimulation of the reticular formation.
Br J Anaesth
2003
;
91
:
233
8
51.
Workman
AD
,
Charvet
CJ
,
Clancy
B
,
Darlington
RB
,
Finlay
BL
:
Modeling transformations of neurodevelopmental sequences across mammalian species.
J Neurosci
2013
;
33
:
7368
83
52.
Schwaller
F
,
Fitzgerald
M
:
The consequences of pain in early life: Injury-induced plasticity in developing pain pathways.
Eur J Neurosci
2014
;
39
:
344
52
53.
Laprairie
JL
,
Johns
ME
,
Murphy
AZ
:
Preemptive morphine analgesia attenuates the long-term consequences of neonatal inflammation in male and female rats.
Pediatr Res
2008
;
64
:
625
30
54.
Sternberg
WF
,
Scorr
L
,
Smith
LD
,
Ridgway
CG
,
Stout
M
:
Long-term effects of neonatal surgery on adulthood pain behavior.
Pain
2005
;
113
:
347
53
55.
Walker
SM
,
Fitzgerald
M
,
Hathway
GJ
:
Surgical injury in the neonatal rat alters the adult pattern of descending modulation from the rostroventral medulla.
Anesthesiology
2015
;
122
:
1391
400