J Neurosurg 72:252-261, 1990

Possible protective effect of endogenous opioids in traumatic brain injury RONALD L. HAYES, PH.D., BRUCE G. LYETH, PH.D., LARRY W. JENKINS, PH.D., RICHARD ZIMMERMAN, M.D., TRACY K. MCINTOSH, PH.D., GuY L. CLIFTON, M.D., AND HAROLD F. YOUNG, M.D.

Division of Neurosurgery, Richard Roland Reynolds Neurosurgical Research Laboratories, Department of Surgery, Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia ~" Naloxone (0.1, 1.0, or 20.0 mg/kg), morphine (1.0 or 10.0 mg/kg), or saline was administered systemically intraperitoneally to rats 15 minutes prior to moderate fluid-percussion brain injury. The effects of the drugs were measured on systemic physiological, neurological, and body-weight responses to injury. The animals were trained prior to injury and were assessed for 10 days after injury on body-weight responses and neurological endpoints. Low doses of naloxone (0. I or 1.0 mg/kg) significantly exacerbated neurological deficits associated with injury. Morphine (10.0 mg/kg) significantly reduced neurological deficits associated with injury. The drugs had no effect on neurological measures or body weight in sham-injured animals. Drug treatments did not significantly alter systemic physiological responses to injury. Data from these experiments suggest the involvement of endogenous opioids in at least some components of neurological deficits following traumatic brain injury and suggest the possibility that at least some classes of endogenous opioids may protect against long-term neurological deficits produced by fluid-percussion injury to the rat. KEY WORDS opioids percussion injury 9 rat 9

p

naloxone

REVIOUS research has indicated that traumatic brain injury (TBI) increases levels of endogenous opioids. Naloxone, a nonspecific opioid antagonist, significantly reverses the hypotension and reduction in pulse pressure following fluid-percussion injury in cats. 26 There are also recent clinical reports of increased t3-endorphin levels in the cerebrospinal fluid of head-injured patients. 8~ Other laboratory studies have indicated that dynorphin A-immunoreactivity (but not leucine-enkephalin or r immunoreactivity) increased in the brain regions o f cats with TBI, showing pronounced histopathology 51 and reduced cerebral blood flow (CBF). 5~ Administration of an opioid kappa-receptor antagonist improved mean arterial blood pressures, regional CBF, and survival in cats following experimental TBI. 5~ Conversely, administration o f a k a p p a agonist after injury worsened neurological outcome and mortality in rats following experimental TBI. 52 These final observations were interpreted as suggesting that, through actions at k a p p a receptors, dynorphin may contribute to the pathophysiology of head t r a u m a associated with secondary disturbances in CBF.

952

9 morphine

traumatic brain injury

9 fluid-

A n u m b e r of additional observations have been interpreted as evidence that secondary or delayed injury following t r a u m a to the central nervous system (CNS) may be due to the release of endogenous opioid "autodestructive" factors. Naloxone has been reported to improve neurological outcome and physiological responses, including spinal cord blood flow, after experimental spinal injury in a variety of species. 19'2~ Other reports have suggested that naloxone has therapeutic efficacy in certain models of cerebral ischemia.4,17,18,32 Considerable caution must be exercised in attributing the pathophysiology of TBI to opioid mechanisms. Although m u c h research on opioid mechanisms of brain injury has emphasized their possible role in vascular changes, clinical studies of TBI have not provided evidence that ischemia is present in patients who survive mild, moderate, or even severe head injury when adequate cerebral perfusion pressures are maintained. A possible consideration is that ischemia m a y occur immediately after injury before flow measurements are made at the hospital. However, ischemic flow reductions have not been observed in at least some experi-

J. Neurosurg. / Volume 72/February, 1990

Protective opioid mechanisms in brain injury mental models of TBI. 13.14.36.43Furthermore, in contrast to findings in cats and rabbits, naloxone does not seem to reliably improve neurological outcome in the rat following spinal cord i n j u r y . 46 Thus, specific endogenous opioid processes do not appear to be universal among different models of spinal cord injury in different species. Additionally, any contribution by dynorphin A to spinal cord injury in the rat may be through nonopioid actions. 46 Recent work from our laboratory (in conjunction with observations by others) argues that certain endogenous opioids may reduce rather than exacerbate the pathophysiological consequences of TBI. Both TBI and seizures may share c o m m o n excitotoxic mechanisms related to excessive release of acetylcholine and excitatory amino acids 27'47'49 (see reviews of this topic29'35'48). Since there is substantial evidence that certain exogenous and endogenous opioids may act as anticonvulsants, 31,72it is possible that some opioids may attenuate excitotoxic processes contributing to long-term deficits following TBI. Because of these observations, we chose to evaluate the effects of naloxone hydrochloride and morphine sulfate on neurological and systemic physiological responses to TBI in the rat. These studies sought to determine: 1) whether pretreatment with naloxone reduced or exacerbated long-term neurological deficits; 2) the most effective dose of naloxone; 3) if naloxone alters systemic physiological responses to injury; and 4) the most effective dose o f morphine if the neurological effects of pretreatment with varying doses of morphine systemically differed from the effects of naloxone. Materials and Methods

Drug Choice Rationale Naloxone has antagonistic actions at more than one receptor subtype, acting preferentially at mu, delta, and kappa receptors? 8 Thus, doses were selected ranging from a dose producing predominant antagonism of mu receptors (0.1 mg/kg) to a dose presumably antagonizing mu, delta, and kappa receptors (20 mg/kg). Since preliminary data with naloxone indicated that mu receptor antagonism could exacerbate neurological deficits, the relatively mu-selective agonist, morphine sulfate, was also evaluated in our model.

Surgical Preparation and Experimental Injury Twenty-four hours prior to trauma, male SpragueDawley rats, weighing 300 to 350 gm each, were surgically prepared for fluid-percussion brain injury under intraperitoneal sodium pentobarbital anesthesia (54 mg/kg) by attaching a hollow injury tube (a modified Leur-loc syringe hub, 2.6 m m inside diameter) epidurally over a trephined hole on the sagittal suture midway between the lambda and bregma. Animals received moderate levels of injury associated with transient neurological suppression as well as more enduring motor deficits. Since preliminary data indicated that naloxone

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may exacerbate and morphine reduce neurological deficits following TBI, mean injury ranges in naloxone (2.20 ___ 0.05 atm) and morphine (2.35 _ 0.05 atm) studies were selected to maximize detection of drug effects. A fluid-percussion device was used to produce experimental brain injury and is described in greater detail elsewhere. 15'66 This device consists of a Plexiglas cylindrical reservoir, 60 cm long and 4.5 cm in diameter, bounded at one end by a rubber-covered Plexiglas piston mounted on O-rings. The opposite end of the reservoir is fitted with a transducer housing ending with a Leur-loc fitting (2.6 m m inside diameter). The entire system is filled with isotonic saline at 37~ Injury is induced by the descent of a metal pendulum striking the piston, thereby injecting a small volume of saline epidurally into the closed cranial cavity and producing brief displacement and deformation o f neural tissue. The resulting pressure pulse is measured in atmospheres (atm) by an extracranial transducer and recorded on a storage oscilloscope.* Fluid percussion has been widely used by a number of laboratories and produces many features of brain injury resembling pathophysiological responses seen in human head injury. These include: brief pressure transients similar to those recorded in h u m a n cadaver skulls during sudden impact; 44'45neurological suppression and neurological signs 15"z8 resembling unconsciousness in humans; 68 reduction or abolition of cerebrovascular responsiveness to changes in pCO2 and pO2; 43'64'77 lOSS of pressure and autoregulation; 42 and immediate and late developing increases in intracranial pressure. 6~ Fluid-percussion injury in the rat 15 produces neurological signs of areflexia, flaccid coma, and stupor similar to those reported in other species and man. Additionally, this model produces motor deficits lasting as long as 7 days following moderate levels of injury. Moderate injury in the rat also produces acute hypertension, bradycardia, increased plasma glucose levels, and suppression of electroencephalographic amplitude and brain-stem auditory evoked potentials in the absence o f significant histopathological alterations. As in the cat model of fluid-percussion i n j u r y , 13'36'43 n o evidence of ischemia has been observed in rats receiving moderate levels of injury in these experiments. 14

Neurological Evaluations In neurological studies, animals were anesthetized with methoxyflurane 5 minutes prior to injury. Following injury, ventilatory support (room air) was provided as needed by connecting the rat to a rodent respirator with a 60-cc syringe securely placed over the nose and mouth. Transient Neurological Suppression. The duration *Transducer, Model PA 85-100, manufactured by Statham, Oxnard, California; oscilloscope, Model 5111, manufactured by Tektronix Inc., Beaverton, Oregon. 253

R. L. Hayes, et al. o f suppression of various reflexes and responses immediately after injury was measured using a battery" of tests which have been described in detail elsewhere. ~.47 A composite score for traumatic unconsciousness was produced by averaging the duration of suppression of the pinna, corneal, paw-withdrawal, tail-flexion, and righting reflexes as well as escape, head support, and spontaneous locomotion responses. The duration of required ventilatory support and incidence of acute mortality (death occurring < 60 minutes after injury) and cumulative mortality (death occurring < 10 days after injury) were noted. The presence o f convulsions was recorded. Any surviving rats that convulsed (an u n c o m m o n feature of this model ~5) were excluded from these studies. Long-Term Neurological Deficits. Persistent neurological disturbances of balance and dizziness are comm o n features o f moderate human head injury. 62 Components o f gross vestibulomotor function were assessed in these rats using a beam-balancing task. This consisted o f placing the animal on a suspended narrow wooden beam (1.0 cm wide, 1.0 m above the floor) and measuring the duration (up to 60 seconds) that it remained on the beam. Training consisted o f three trials approximately 1 hour prior to trauma, which also served as preinjury baseline measures. Persistent m o t o r retardation and disturbances in fine m o t o r skills are c o m m o n features o f moderate human head injury. 4~ Components o f fine m o t o r coordination in the rat were assessed using a beam-walking task similar to that used by Feeney and coworkers. 2~ Two days prior to injury, rats were trained to escape a bright light and loud white noise by traversing a narrow wooden beam (2.0 • 100.0 cm) to enter a darkened goal box at the opposite end o f the beam. During training and testing, the animals were placed at one end of the beam close to the source of light and noise. The noise and light were terminated when the rat entered the goal box at the opposite end o f the beam. Four steel pegs (3.0 m m in diameter and 4.0 cm high) were placed in an alternating sequence along the beam to increase the difficulty o f the task. Performance was assessed by measuring the period o f time for the animal to traverse the beam. Data for each daily session consisted of the mean o f three trials.

Systemic Physiological Evaluations Rats were initially anesthetized with methoxyflurane followed by a mixture o f 4% halothane, 70% NO2, and 30% 02. The animals were intubated, paralyzed with intraperitoneal curare (0.3 ml), and mechanically ventilated.t They were then maintained on a mixture of 1.5% halothane, 70% NO2, and 30% 02. The left femoral artery was cannulated with polyethylene (PE 50) tubing for monitoring blood pressure and sampling t Animal respirator, Model 680, manufactured by Harvard Apparatus, Cleveland, Ohio. 284

blood. Blood gases were analyzed using a pH/blood gas analyzer, and plasma glucose levels were determined by means of the oxygen rate method.z~ All surgical wounds were infused with a 1% lidocaine hydrochloride solution during surgery and throughout the experiment. Respiration was adjusted throughout the experiment to maintain normal pCO2 and p02 values.

Experimental Protocols Experiment L Experiment I was designed to assess

the effects o f three doses of naloxone administered prior to injury on transient neurological suppression and long-term neurological deficits associated with moderate fluid-percussion injury. For this experiment, the rats were trained on the beam-walking and beam-balancing tasks and surgically prepared for fluid-percussion injury 24 hours prior to brain injury. The animals were assigned by latin-square design to one of four treatment groups (10 rats per group, each surviving at least 24 hours): naloxone (0.1, 1.0, or 20.0 mg/kg) or saline (equal volume). Drugs were administered intraperitoneally 15 minutes prior to injury. Investigators were blind to both the drug and dosage administered. The animals were anesthetized with methoxyflurane 5 minutes prior to injury and were assessed daily for 10 days after injury. Additional animals (five per group) received naloxone (0.1, 1.0, or 20.0 mg/kg) or saline (equal volume) t 5 minutes prior to sham injury. These animals were trained in the same m a n n e r as the injured animals and were assessed for 3 days after sham injury. Experiment IL Experiment II was designed to assess the effects o f two doses of morphine administered prior to injury on transient neurological suppression and long-term neurological deficits associated with moderate fluid-percussion injury. For this experiment the rats were trained and surgically prepared for fluid percussion as in Experiment I. The animals were assigned by latinsquare design to one of three treatment groups (nine to 10 survivors per group): morphine (1.0, or 10.0 mg]kg) or saline (equal volume). Drugs were administered intraperitoneally 15 minutes prior to injury. Investigators were blind to both the drug and dosage administered. The animals were anesthetized with methoxyflurane 5 minutes prior to injury and were assessed daily for 10 days after injury. Additional animals (five per group) received morphine (1.0 or 10.0 mg/kg) or saline (equal volume) 15 minutes prior to sham injury. These animals were trained in the same manner as the injured animals and were assessed for 3 days after sham injury. Experiment III. Experiment III was designed to assess the effects o f naloxone and morphine on systemic {pH/blood gas analyzer, Model 158, manufactured by Coming Glass Works, Coming, New York; glucose analyzer, Model 2, manufactured by Beckman Instruments, Inc., Fullerton, California.

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Protective opioid mechanisms in brain injury physiological responses to fluid-percussion injury. For this experiment the rats were assigned to one of four treatment groups (five per group): naloxone (0.1 or 20.0 mg/kg), morphine (10.0 mg/kg), or saline vehicle (equal volume). Drugs were administered intraperitoneally 15 minutes prior to injury. The animals were monitored for 50 minutes after injury.

Histopathological Study Previous studies have extensively characterized the histopathology for this brain injury range. ~5To confirm the homogeneity of injury, selected animals were prepared for light microscopic examination. The animals were perfused transcardially with 10% buffered neutral formalin 10 days after injury. Their brains were removed, dehydrated in ascending grades of ethanol, cleared, and embedded in paraffin. The tissue was cut into 8-t* sagittal sections, mounted, and stained with hematoxylin and eosin (H & E). Statistical Analysis Data for long-term neurological and cardiovascular measures were analyzed using a repeated measures analysis of variance (drug x time) followed by Duncan multiple-range post hoc tests when appropriate. Data for ventilatory support and transient neurological suppression were analyzed using analysis of variance followed by Tukey post hoc tests when appropriate. Nonparametric mortality data were analyzed using chisquared testing. Significance levels were set at p < 0.05. Means are expressed _+ standard error of the mean. Results Experiment I The duration of transient neurological suppression was not significantly different among the groups. The mean composite durations of suppression for each group were: saline 8.2 + 2.0 minutes; naloxone, 0.1 mg/kg, 12.6 _+ 1.7 minutes; naloxone, 1.0 mg/kg, 8.8 -+ 1.3 minutes; and naloxone, 20.0 mg/kg, 8.3 + 1.5 minutes. None of the groups differed significantly from the others in baseline preinjury assessments of behavior used to measure long-term deficits. Saline-treated rats exhibited deficits in beam-walking on Day 1 after injury compared to preinjury scores. Rats pretreated with naloxone, either 0.1 or 1.0 mg/kg, showed significant (p < 0.05) deficits in beam-walking compared to preinjury scores for Days 1 to 9 and Days I to 8 after injury, respectively (Fig. 1A). The saline-treated rats exhibited a tendency for decreased beam-balancing performance compared to preinjury scores; however, this decrement was not statistically significant (p < 0.10). The rats treated with naloxone, 0.1 and 1.0 mg/kg, showed significant (p < 0.05) beam-balancing deficits on Day 1 after injury compared to their preinjury performance (Fig. 1B). The body weight of saline-treated rats dropped sigJ. Neurosurg. / Volume 72/February, 1990

FIG. 1. Dose-response effects of naloxone treatment on (A) beam-walking latency, (B) beam-balancing duration, and (C) changes in body weight following moderate fluid-percussion brain injury in rats. Drugs were administered intraperitoneally 15 minutes prior to injury. The data represent means --. standard error of the means prior to (Day 0) and at various days after injury. Asterisks: p < 0.05 versus Day 0 values. Both the 0.1- and 1.0-mg/kg doses of naloxone significantly increased the duration of these deficits.

nificantly (p < 0.05) below preinjury values on Days 3 and 4 after injury. The rats pretreated with 0.1- and 1.0-mg/kg doses of naloxone showed significant loss of body weight for Days 1 to 10 and Days 1 to 7 after injury, respectively. The animals pretreated with naloxone, 20 mg/kg, showed no significant postinjury loss in body weight (Fig. IC). Administration of naloxone (0.1, 1.0, or 20.0 mg/kg) or saline had no effect on long-term neurological performance after sham injury (Fig. 2).

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R. L. Hayes, et al.

FIG. 2. The effects of naloxone and morphine treatment on (A) beam-walking latency, (B) beam-balancing duration, and (C) changes in body weight following sham brain injury in rats. Drugs were administered intraperitoneally 15 minutes before sham injury. The data represent means _ standard error of the means prior to (Day 0) and at various days after sham injury. These drugs did not significantly affect these measures in sham-injured rats.

FIG. 3. The effects of morphine treatment on (A) beamwalking latency, (B) beam-balancing duration, and (C) changes in body weight following moderate fluid-percussion brain injury in rats. Drugs were administered intraperitoneally 15 minutes prior to injury. The data represent means +standard error of the means prior to (Day 0) and at various days after injury. Asterisks: p < 0.05 versus Day 0 values. Morphine (10.0 mg/kg) provided significant reductions in the durations of these deficits.

Treatment with naloxone did not influence the duration of ventilatory support required to compensate for apnea (p > 0.90). The mean durations of ventilatory support for animals receiving either saline or 0.1, 1.0, or 20.0 mg/kg of naloxone were 13.8 ___4.6, 13.8 _ 5.1, 14.2 + 5.4, and 18.5 __+6.8 seconds, respectively. Treatment with naloxone had no statistically reliable effect on the acute mortality rate (within the first 60 minutes after injury) (p > 0.75) or the cumulative mortality rate over 10 days (p > 0.45). The cumulative mortality rate over the 10-day observation period for

each treatment group was as follows: saline 44% (seven of 16 rats); naloxone, 0.1 mg/kg, 47% (nine of 19 rats); naloxone, 1.0 mg/kg, 23% (three of 13 rats); and naloxone, 20.0 mg/kg, 31% (four of 13 rats).

256

Experiment H The duration of transient neurological suppression was not significantly different among the groups. The mean composite durations of suppression for each group were: saline, 15.1 __+2.6 minutes; morphine, 1.0 mg/kg, 18.8 _ 3.7 minutes; and morphine, 10.0 mg/ J. Neurosurg. / Volume 72/February, 1990

Protective opioid mechanisms in brain injury TABLE 1 Arterial plasma glucose levels in the four treatment groups* Treatment

Pretrauma

Posttrauma Level

Level

5 Min 30 Min 50 Min saline 158.5___10.2 179.3_+38.5 180.3+30.4 140.0___26.5 naloxone, 0.1 184.8+19.9 194.2+20.4 167.3+7.5 213.5___33.5 mg/kg naloxone, 20.0 156.8+12.3 197.6-+30.3 192.5__+25.9 122.8+18.5 mg/kg morphine, 10.0 131.4-,10.1 122.8+5.5 125.8+7.6 150.5___12.6 mg/kg * Values are means _ standard error of the means (mg/100 ml). There were five rats in each group. FIG. 4. The effects of naloxone and morphine on mean arterial blood pressure following moderate fluid-percussion injury in rats. The data represent means + standard error of the means prior to and for 50 minutes after injury. Drugs were administered intraperitoneally 15 minutes prior to injury. Asterisks: p < 0.05 versus Day 0 values. These drugs did not have a significant effect on cardiovascular responses to injury.

kg, 20.4 _+ 2.8 minutes. Morphine treatment (10.0 mg/ kg) significantly increased the duration of escape response in injured animals (morphine 45.0 _+ 6.2 minutes vs. saline 18.8 + 4.6 minutes) and sham-injured animals (morphine 13.6 _+ 11.6 minutes vs. saline 0.7 _+ 0.1 minutes). N o n e of the groups differed significantly from the others in baseline preinjury assessments of behavior used to measure long-term deficits. The saline-treated rats exhibited significant beam-walking deficits compared to preinjury baseline on Days 1 to 6, 8, and 10 after injury. The rats treated with morphine, 1.0 mg/ kg, exhibited significant deficits on Days l, 2, and 4 after injury. The animals treated with a 10.0-mg/kg dose of morphine exhibited significant deficits only on Days 1 to 3 after injury (Fig. 3A). The rats treated with saline exhibited significant beam-balancing deficits on Day 1 after injury compared to preinjury baselines. The group treated with a 1.0mg/kg dose of morphine exhibited significant deficits on Days 1 and 2 after injury. The rats treated with morphine, 10.0 mg/kg, did not exhibit significant beambalancing deficits after injury (Fig. 3B). The rats treated with saline or morphine, 1.0 mg/kg, lost significant body weight over the 10-day observation period after injury and never recovered to preinjury weights. Rats pretreated with 10.0 mg/kg of morphine did not experience significant body weight loss after injury and weighed significantly more than salinetreated rats in Days 3 to 10 after injury (Fig. 3C). Administration o f morphine (1.0 or 10.0 mg/kg) had no effect on long-term neurological performance after sham injury (Fig. 2). Treatment with morphine did not influence the duJ. Neurosurg. / Volume 72/February, 1990

ration of ventilatory support required to compensate for apnea (p > 0.85). The m e a n durations of ventilatory support for animals receiving either saline or morphine, 1.0 or 10.0 mg/kg, were 15.1 _+ 4.0, 18.7 _+ 6.0, and 18.0 _+ 6.3 seconds, respectively. Treatment with morphine had no statistically reliable effect on the acute mortality rate (within the first 60 minutes after injury) (p < 0.08) or the cumulative mortality rate over l0 days (< 0.06). The cumulative mortality rate over the 10-day observation period was: saline 33% (four of 12 rats); morphine 1.0 mg/kg, 63% (12 of 19 rats); and morphine, 10.0 mg/kg, 23% (three of 13 rats). Experiment III Mean arterial blood pressure significantly increased (p < 0.05) in all treatment groups following injury (Fig. 4). Hypertension peaked within 15 seconds after injury and returned to baseline by 30 minutes after injury. Plasma glucose levels changed very little after injury in all drug treatment groups (Table 1). There was a nonsignificant trend for plasma glucose levels to increase at 5 and 30 minutes after injury for the saline and naloxone treatment groups. Blood gas values were maintained within normal ranges 3 for all groups throughout the experiment. Histopathological Study. The evaluation of H & Estained parasagittal sections of each rat brain revealed no apparent structural abnormalities such as macro- or microintraparenchymal hemorrhages, gliosis, or overt neuronal cell loss. Discussion

Data from our laboratory experiments suggest the involvement o f endogenous opioids in at least some components o f long-term deficits following traumatic brain injury. In Experiment I, single doses o f naloxone, either 0.1 or 1.0 mg/kg, significantly exacerbated certain long-term neurological deficits. Conversely, Experim e n t II showed that pretreatment with 1.0- or 10.0mg/kg doses of morphine significantly reduced certain o f these deficits. Considered together, these data suggest 257

R. L. Hayes, et al. the possibility that at least some classes of endogenous opioids may protect against long-term deficits produced by fluid-percussion injury to the rat. We have hypothesized that TBI produces widespread depolarization of neurons and excessive release of excitatory neurotransmitters and/or neuromodulators, including acetylcholine (ACh) and glutamate, thereby resulting in excitotoxic effects of postsynaptic neuronal e l e m e n t s , z7'29'47'48 While these postsynaptic excitotoxic changes may not result in the overt cell death frequently observed following seizure activity, ~ they could contribute to long-term neuronal dysfunction underlying persistent neurological deficits following TBI. There is considerable evidence that cholinergic57'76and glutamatergic' 1.61 processes can contribute to excitotoxic neural injury produced by seizures, perhaps by interactions with postsynaptic muscarinic cholinergic or N-methylD-aspartate (NMDA) receptors. We have also shown that blockade of muscarinic47 and NMDA 27 receptors protects against long-term neurological deficits following TBI to the rat. Much evidence suggests that certain classes of opioids might have a protective effect on seizure activity. Although some research has suggested that opioids are convulsive,s~ numerous studies clearly indicate that exogenously administered opioid alkaloids and peptides protect against experimentally induced seizures. ~'12 More recently, researchers have postulated that central opioid systems may function postictally as anticonvulsants. 3~'72 Consistently, naloxone has been shown to facilitate kindlingY Endogenous opioid systems have been implicated in the rise in seizure thresholds after seizures induced by electroconvulsive shock69 and in postictal inhibition of seizures in kindled rats. 37 An endogenous anticonvulsant opioid peptide has been detected in rat cerebrospinal fluid after generalized seizures. 71,73 More generally, the protective effects of endogenous opioids may be related to their ability to attenuate excitotoxic processes acting as inhibitory modulators of neural functioning. Both adenosine ~6and gamma-aminobutyric acid (GABA)ergic compounds 53 are anticonvulsant agents, and recent research has suggested that increased inhibitory tone produced by an adenosine analogue67 or by GABAergic agents65 can attenuate experimental ischemic brain damage, a process to which glutamatergic excitotoxic mechanisms presumably contribute. H'rt Opioids have widespread inhibitory effects on CNS function. 56 Opioid agonists can reduce neuronal excitation by amino a c i d s . 34 In vivo studies indicate that both methadone and morphine can reduce NMDA receptor-mediated neurotoxicity, although possibly not by actions at opioid receptors. 9 Related opioid sigma agonists, which also may act directly at NMDA receptors, 33 have been reported to have inhibitory antiexcitotoxic properties. Dextrorphan 1~and other sigma agonists6 can depress neuronal excitation by amino acids, and dextrorphan is an anticonvulsant agent22 which can reduce glutamate neurotoxicity.7 The sigma 258

agonist and precursor of dextrorphan, dextromethorphan, is an anticonvulsant drug TM reported to reduce glutamate neurotoxicity7 and hypoxic-ischemic~9 and ischemic cell i n j u r y . 24 Opioids may also reduce cholinergic activity. Endogenous opioid peptides can attenuate release of ACh, 2"38 and dextrorphan reduces neuronal excitation by ACh.~~ Since naloxone has antagonistic actions at more than one opioid receptor subtype, ~sour studies provided data that only indirectly suggested the involvement of specific classes of opioid receptors. The significant exacerbation of long-term neurological deficits by a 0.1-mg/ kg dose of naloxone suggests that binding of an endogenous opioid to the mu receptor may be protective. The observation is consistent with the protective effects of morphine, which binds predominantly to mu receptors. In contrast, most evidence indicates that endogenous opioid systems, acting postictally as anticonvulsant agents, may act through delta receptors. 3~'72 However, since both mu and delta receptor systems may mediate opioid anticonvulsant properties, investigators have speculated that both receptor subtypes may be involved, possibly by a functional coupling between mu and delta binding sites. 3~ The highest dose of naloxone (20 mg/ kg), presumably also binding to kappa receptors, did not exacerbate long-term deficits. In fact, a nonsignificant trend of reduced deficits was observed. Thus, it is conceivable that, consistent with previous research, 5~a specific kappa antagonist may provide neurological protection following TBI. However, there are also reports that kappa agonists are anticonvulsant agents7~ and can produce some excitotoxic protection?9 Current studies in our laboratories employing more specific opioid receptor ligands are addressing these issues. The molecular mechanisms by which endogenous opioid-receptor interactions could attenuate the pathophysiology of TBI are not yet understood. In view of data indicating that increased intracellular Ca ++ could contribute to excitotoxic injury,8 descriptions of the abilities of opioids to alter Ca++-dependent processes may be critical to molecular explanations of opioid influences on TBI. Acute opioid treatment reduces interactions of Ca +§ with nerve membranes by decreasing Ca § content, binding, Ca++/Mg §247adenosine triphosphatase, and Ca++ uptake into synaptosomal endoplasmic reticulum, and voltage-dependent Ca ++ influx via Ca ++ channels. Opioid-induced reductions of intracellular Ca++ could have a variety of effects including reduced excitation of postsynaptic neurons. For example, binding of opioids to their presynaptic receptors could reduce transmitter release by decreasing Ca§ influx into neural tissue. This topic is reviewed elsewhere. TM Neither morphine nor naloxone significantly influenced the mortality rate following TBI. We have previously reported that pretreatment with both NMDA 27 and muscarinic47 receptor antagonists could reduce the mortality rate and the incidence of long-term deficits. The data presented here suggest that, in contrast to ACh J. Neurosurg. / Volume 72/February, 1990

Protective opioid mechanisms in brain injury and excitatory amino acids, opioid mechanisms may not significantly contribute to death. Although the systemic administration of drugs did not allow us to make inferences about specific CNS sites of drug actions, several observations suggest that opioid modification o f the pathophysiology o f TBI is attributable to direct effects on neurons. In these studies, neither naloxone nor morphine affected the hypertension or the elevated plasma glucose levels following TBI, suggesting that changes in sympathoadrenal responses did not contribute to neurological outcome following drug treatment. Importantly, apnea is reliably associated with death following TBI.L5 Although opioids can influence respirator3, function, including changes in respiration following electroconvulsive shock, ~ neither morphine nor naloxone altered the duration of respiratory depression following TBI. Since the animals in this study were also provided ventilatory support, it is unlikely that reduced oxygen availability contributed to neurological deficits in both control and drug-treated rats. Although opioids have well-documented effects on food intake, 5a postinjury changes in body weight associated with naloxone or morphine pretreatment were probably not related to nonspecific drug effects since neither drug had prolonged effects on the body weight of sham-injured rats. Finally, it seems unlikely that opioid effects in our model were related to changes in CBF. Previous studies in the cat detected no significant reductions in CBF following T B I . 13'36"43 Preliminary studies in our laboratory have revealed no evidence of ischemia following TBI in the rat.14 Naloxone or morphine had no effect on the composite measures o f transient neurological suppression in injured and sham-injured rats. Not surprisingly, morphine treatment (I0 mg/kg) significantly increased the duration of suppression of the nociceptive escape-response measure in both injured and sham-injured animals. Previous work in our laboratories has provided much evidence that increased activity in cholinergic pontine inhibitory centers, rather than excitotoxic changes, contributes to transient neurological suppression following TBI. This topic is reviewed elsewhere. 29'3~ For example, an N M D A receptor antagonist can reduce long-term deficits without affecting transient neurological suppression, z7 The present experiments provide additional evidence clearly dissociating mechanisms mediating transient and long-term neurological alterations. While naloxone (0.1 mg/kg) increased and morphine (10.0 mg/kg) reduced long-term deficits, these drugs had no effect on composite measures of transient neurological suppression. Physiological experiments indicated that the effects o f drug pretreatment on neurological outcome were not related to changes in cardiovascular responses or alteration in plasma glucose levels, both of which are strongly influenced by sympathically mediated responses to trauma. Previous research on TBI has shown that other drugs can attenuate neurological deficits without altering the sympathoadrenal response to in,Jr.Neurosurg. / Volume 72/February, 1990

j u r y . 47 Other studies indicate that CNS injury not in-

volving energy failure may not be influenced by hyperglycemia. Levels o f hyperglycemia that exacerbate hypoxic ischemic CA 1 damage does not exacerbate status epilepticus CA 1 changes induced by kainic acid. 63 Thus, it appears likely that the neurological consequences o f TBI are not strongly influenced by the physiological factors measured in this study. These studies have provided evidence that certain endogenous opioid substances may attenuate pathophysiological responses to experimental TBI. Although the studies reported here employed drug pretreatment with bolus injections, these observations have implications for the treatment o f h u m a n head injury. First, any proposal to treat h u m a n head injury with opioid antagonists should proceed cautiously in view o f the possibility that blockade o f certain opioid receptors could exacerbate the injury. Second, the possible identification of endogenous protective substances could provide important insights into the development o f improved pharmacological agents to treat head injury. Acknowledgments

The authors gratefully acknowledge the excellent editorial assistance of Fay Akers. This manuscript is dedicated to the memory of Rena Chapouris, whose competence and industry made this research possible. References

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Manuscript received May 19, 1989. This research was supported by Grant H133B80029 from the National Institute on Disability and Rehabilitation Research, the U.S. Department of Education, and National Institutes of Health Grants NS 21458 and NS 12587. Address reprint requests to: Ronald L. Hayes, Ph.D., Medical College of Virginia, Box 693, MCV Station, Richmond, Virginia 22298-0693.

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