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PEDIATRIC ANESTHESIOLOGY

General Anesthetics Induce Apoptotic Neurodegeneration in the Neonatal Rat Spinal Cord

Robert D. Sanders, BSc, MBBS^*, Jing Xu, MD, Yi Shu, BSc^*, Antonio Fidalgo, MSc^*, Daqing Ma, MD, PhD^*, and Mervyn Maze, MB ChB, FRCA, FRCP, FMedSci^*

From the *Departments of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, Chelsea & Westerminster Hospital, London; and Department of Anesthesiology, Gongli Hospital, Pudong, Shanghai, China.

Address correspondence and reprint requests to Dr. Daqing Ma, Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London SW10 9NH. Address e-mail to d.ma{at}imperial.ac.uk.

Abstract

BACKGROUND: Exposure to anesthetics triggers apoptotic neurodegeneration in the neonatal rat brain; whether neuronal apoptosis also occurs in the spinal cord, a crucial target for analgesic and anesthetic drugs, is unknown.

METHODS: We exposed 7-day-old rats were exposed to air or 75% nitrous oxide + 0.75% isoflurane in oxygen for 6 h (n = 19 per group). Caspase-3 immunoreactivity was evaluated in the lumbar spinal cord at the end of the gas exposure (n = 3 per group). Developmental nociceptive responses were tested using tail flick latencies on postnatal days 8, 15, and 30 (n = 3 per group). Motor responses were evaluated using the rotarod on postnatal day 30 (n = 7 per group).

RESULTS: Isoflurane plus nitrous oxide increased the numbers of caspase-3 positive neurons in the spinal cord (P < 0.01). Despite a preponderance of the injury in the ventral horn of the spinal cord, motor impairment did not occur (P > 0.05). No functional effect on nociception was observed at the three developmental stages tested (P > 0.05).

CONCLUSIONS: Anesthesia induces apoptosis in the neonatal rat spinal cord; however, the functional consequences of this injury, if any, remain obscure. Neither motor nor nociceptive responses were affected by anesthetic treatment. Nonetheless, further investigation is required as regional anesthetic techniques may also trigger neuroapoptosis in the spinal cord with unknown potency.

Anesthesia induces neuroapoptosis throughout forebrain structures in the neonatal rat brain, producing prolonged impairment of neurocognition1–5 and leading to concerns over the safety of anesthetic exposure to young humans up to 3-yr-of-age.6 The mechanism of this injury is still largely unknown, though current hypotheses include the suggestion that inhibition of synaptic activity during the critical period of synaptogenesis leads to a reduction in trophic signaling triggering the nerve cell to deem itself redundant, and thus provoking apoptotic cellular death.2,4 However, the extent of the spatial distribution of this injury in the central nervous system is still unknown.

We therefore embarked on a study focusing on the spinal cord as a possible site of injury secondary to anesthetic exposure to isoflurane and nitrous oxide; a combination that provokes robust neuroapoptosis in the neonatal rat brain.1,3,4 Our null hypothesis was that anesthetic treatment would not induce apoptosis in the neonatal rat spinal cord. Having established an anesthetic-induced morphological injury, we investigated whether a functional correlate could be obtained. We assessed nociception, as nociceptive processing involves both the ventral and dorsal horns in the neonate and therefore injury in either horn may affect this functional modality.7–9 Motor responses were also formally evaluated to confirm a previous report, suggesting that they were not affected by neonatal anesthetic treatment.1

METHODS

The study protocol was approved by the Home Office (UK) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986. Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of nitrous oxide (75%) + isoflurane (0.75%) in oxygen (25%) or air in a temperature-controlled chamber (n = 19 per group). Littermate pups were randomly assigned between groups. The gases were delivered by anesthetic machine gas concentrations for oxygen, isoflurane, and nitrous oxide were monitored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). Body temperature of the pups was maintained as described previously4 with individual exposure chambers partially submerged in a water bath, and the water temperature adjusted to obtain a desired brain temperature (37°C) as measured with a telemetry temperature monitoring system (VitalView; Mini-Mitter, Sunriver, OR).

Three animals per group were then killed (with sodium pentobarbital 100 mg/kg by intraperitoneal injection) at the end of gas exposure, perfused transcardially with heparinized saline followed by paraformaldehyde 4% in 0.1 M buffer. After removal of the spinal cord, by pneumatic injection it was stored overnight at 4°C in paraformaldehyde and then transferred to sucrose solution (30%) with phosphate buffer and sodium azide 1% and kept refrigerated until the spinal cords were sectioned at the lumbar level (as described previously in Ref.10). The sections were then transferred to a six-well plate containing phosphate buffered saline (PBS). Sections were then preincubated with hydrogen peroxidase 0.3% in methanol for 30 min and then rinsed in PBS before being incubated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2500; New England Biolab, Hitchin, UK) and then washed three times in PBS with Triton 3% at room temperature. Biotinylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, UK) were applied. The sections were again washed in PBS before incubation with 0.02% 3,3'-diaminobenzidine with nickel ammonium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70%–100%) and then mounted (floating section) and covered with a cover slip. The number of caspase-3 immunoreactive neurons were counted in four lumbar spinal cord sections per animal by an investigator blinded to the experimental protocol.

Motor impairment was assessed on postnatal day 30 by placing rats (n = 7 per group) on a rotarod (rotating at 30 rpm) and the time spent on the rod was assessed (maximal latency was 300 s) as we have performed previously.11 Rats were tested three times with a 10-min interval between each assessment and the sum of the three assessments was used for data analysis.

For assessment of tail flick latencies (TFL) (n = 3 per group) at PND 8, 15, and 30, the rats were placed in individual cylindrical plastic restrainers. All experiments were performed between 9 am and 7 pm under normal room light and temperature. The TFL test was measured using the Ugo Basile Analgesia Meter (Biologic Research Apparatus, Varese, Italy). A beam of radiant heat was focused on the underside of the tail on the middle third of the rat’s tail. The latency between the exposure to the radiant heat source and the movement of the tail away from the focused beam was referred to as TFL. A maximum TFL of 10 s was set. Each TFL data point was a mean of four measurements per rat, measured to the nearest 0.1 s.

The results are expressed as median and range (minimum to maximum). The significance level was set a P < 0.05 and Mann–Whitney U-test were used to compare the groups. The ² test was used to compare the differences in numbers of animals who achieved maximum latency on the rotarod with their first trial. Based on our preliminary TFL data, the study of nociception was terminated as power analysis ( = 0.05, β = 0.8) suggested that 146 animals per group (total n = 292) would be required to find a difference between the groups at age PND 8.

RESULTS

Anesthesia with nitrous oxide-isoflurane for 6 h increased the number of caspase-3 positive cells relative to air exposure (P < 0.01) (Fig. 1.). The site expressing the biggest increase in caspase-3 reactive cells appeared to be within the ventral horn of the spinal cord, although dorsal horn neurons were also affected.

View larger version (43K): [in this window] [in a new window]   Figure 1. Apoptotic neurodegeneration induced by 75% Nitrous oxide + 0.75% isoflurane as measured with caspase-3 immunostaining in the lumbar enlargement of spinal cord of 7-day-old neonatal rats (n = 3 per group). A, Air control; B, 75% Nitrous oxide + 0.75% isoflurane in oxygen; C, Mean (mean ± sd; n = 3). Black arrows = caspase 3 positive cells.

In concordance with a previous report,1 motor responses were not affected by neonatal anesthetic treatment [air 300 s (82–300) vs anesthesia 300 s (68–300); P = 0.2]. However, there was a nonsignificant difference between the air and anesthetic groups with respect to their first of the three trials on the rotarod; while 5 of 7 animals achieved maximum latency on the rotarod in the air group on their first trial, only 2 of 7 animals achieved the same in the anesthetic group (P = 0.29).

TFLs did not differ between the groups (n = 3 per group) at PND 8 [air 3.8 s (1.5–9.5) vs anesthesia 3.6 s (2.2–10.0); P = 0.5], PND 15 [air 3.1 s (2.3–8.1) vs anesthesia 3.6 s (2.1–5.2); P = 0.7] or PND 30 [3.8 s (2.3–5.6) vs anesthesia 3.8 s (3.1–5.3); P = 0.9]. After subsequent power analysis, the study of nociceptive responses was terminated.

DISCUSSION

In this study, we have demonstrated that neonatal exposure to 6 h of an isoflurane-nitrous oxide-based anesthetic provokes apoptosis, as evidenced by increased cleaved-caspase 3 immunoreactivity in the neonatal rat spinal cord. However, this injury does not affect either motor or nociceptive function. Nonetheless, the current work demonstrates that the apoptotic injury is of wider distribution in the central nervous system than previously reported.1–6

Consistent with previous studies, we have used caspase-3 immunohistochemistry to identify the apoptotic injury as it is an early marker of cellular apoptosis and has been considered a reliable marker for apoptosis in the context of anesthetic injury6 that correlates with subsequent functional impairment when previously studied in the brain.1 In addition, we previously confirmed the ability of caspase-3 to detect apoptotic neurons with different methods of histology.4,11 The primary limitation of this study remains the small sample sizes involved; however, we stopped immunostaining assessment after three animals per group as the injury was apparent, favoring to focus on finding a functional end-point for the injury. We initially sought a motor end-point due to the preponderance of the injury in the ventral horn of the spinal cord; in concordance with previous results,1 we found motor function was not affected. We collected preliminary data to assess whether nociceptive function may be affected but terminated the study early after our power analysis revealed that if the effect was real it was probably too small to be clinically relevant. On the basis of our data, we would require 146 animals per group to find a difference in TFLs between the groups at PND 8, indicating a very small effect. In addition, we have not reported arterial blood gas or glucose results1,3,12 as these have not been found to be deranged in similar studies and the injury occurs in in vitro settings in which oxygen and glucose are strictly controlled.4 Nonetheless, we consider that we have uncovered some interesting and potentially important findings in this work.

Although we did not observe motor impairment in the rotarod test 30 days after neonatal anesthetic treatment, we found that fewer animals achieved maximum latency in the anesthetic group compared with the air group. This may reflect the previously reported impaired cognitive function in the anesthetic-treated animals as they react poorly to the rotating rod. However, the differences did not achieve significance. Despite increased apoptosis in the ventral horn of the spinal cord, functional motor impairment assessed by the rotarod did not occur.

Altered neurodevelopmental responses to nociception do not occur after exposure to anesthetic drugs assessed by the tail flick test in our study. The test involves a spinal reflex arc, and thus will test nociceptive responses within the spinal cord. This method has been used to detect the altered neurodevelopmental nociceptive processing in the central nervous system induced by the administration of morphine to the neonatal rats.13 Therefore, TFLs are a valid method to assess the potential impact of anesthetics on neurodevelopmental nociception, although we cannot exclude that other aspects of nociception would not be affected by anesthetic exposure, such as tests of hyperalgesia including the formalin, carageenan, or von Frey filament tests.

The observed vulnerability of the ventral horn of the spinal cord to anesthetic injury is in keeping with the ability of anesthetics to suppress neurotransmission in this region.14 There is evidence to suggest that anesthetic depth is important to anesthetic-induced neurotoxicity potentially indicating overlapping mechanisms of action of anesthesia and anesthetic-induced cell death implicated in the prevailing hypothesis for this injury, and as described in the introduction.2 Indeed, escalating doses of isoflurane provoke increasing apoptosis in the brain.1 However, other evidence contradicts this finding, including data showing that xenon and dexmedetomidine can attenuate isoflurane-induced injury despite increasing anesthetic depth.4,15 However, it is possible that both of these drugs inhibit the apoptotic injury while increasing the depth of anesthesia through differing mechanisms. Nonetheless, the data within appears to further a connection between the ability for anesthetics to inhibit neuronal activity and provoke cellular death. The duration of anesthetic exposure is likely to also be important to the injury with early studies using long exposures to anesthetics.1–4 Indeed, we used a long duration of anesthetic exposure (6 h) to survey the spinal cord as a locus for the injury and study the adverse consequences, if any, of the toxicity. Recent data suggest that sub-minimum alveolar concentrations (2%) of isoflurane can induce apoptosis in mice after only 1 h,12 and thus clinically relevant doses and duration of isoflurane may provoke toxicity though this is unconfirmed in primates.

In summary, our finding suggests that general anesthesia has little effect on functional outcomes, but motor and sensory end-points should be studied in future investigations addressing the effects of spinal anesthesia in animal models. Indeed, as regional techniques induce prolonged inhibition of neuronal activity that may last for several hours, investigation of the effects of intrathecal local anesthetic administration is warranted in young animals. This may be especially prudent, as intrathecal lidocaine has been associated with spinal neurotoxicity in older animals.16 However, as the methods used in preclinical studies may be insensitive to the subtle changes induced in the developing nervous system in humans, future clinical trials should consider investigation of sensory and motor responses during development of humans after anesthetic treatment, whether general or regional, in the young.

Footnotes

Accepted for publication March 5, 2008.

Supported by Westminster Medical School Research Trust, London, UK.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sanders, R. D. Articles by Maze, M. Search for Related Content PubMed PubMed Citation Articles by Sanders, R. D. Articles by Maze, M.