Jeffrey R. Kirsch

Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimers disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-D-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis-necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.

Postischemic cerebral blood flow recovery in the female : effect of 17β-estradiol

Female reproductive hormones are considered to be protective agents in atherosclerotic vascular disease and stroke. The present study determined if there are unique cerebrovascular responses in female animals to global cerebral ischemia and if 17β-estradiol is important to postischemic outcome in brain. Three groups of anesthetized, sexually mature rabbits were treated with normotensive four-vessel occlusion (6 min) and 3 h of reperfusion: females chronically instrumented with 17β-estradiol implants (EFEM; n = 8, plasma estradiol level = 365 ± 48 pg/ml), untreated females (FEM; n = 8, estradiol = 13 ± 3 pg/ml), and untreated males (M; n = 8, estradiol < limit of radioimmunoassay). CBF (microspheres) and somatosensory evoked potential (SEP) amplitude were measured during ischemia/reperfusion. Baseline hemispheric blood flow and regional flow distribution were not altered by chronic estradiol treatment. Hemispheric blood flow was equivalently reduced during ischemia in FEM and M (6 ± 1 and 9 ± 2 ml min−1 100 g−1 respectively); however postischemic hyperemia was greater in FEM than M (CBF = 257 ± 27 and 183 ± 27 ml min−1 100 g−1. However, EFEM experienced higher CBF during ischemia (e.g., 13 ± 2 ml min−1 100 g−1) and less hyperemia (134 ± 4 ml min−1 100 g−1 in hemispheres) in numerous brain regions than FEM. CBF at 3 h reperfusion was not different among the groups. Recovery of SEPs was incomplete and similar in all groups. We conclude that chronic exogenous 17β-estradiol treatment increases CBF during global incomplete ischemia and ameliorates postischemic hyperemia in the female animal.

Effect of nitric oxide synthase inhibition on cerebral blood flow and injury volume during focal ischemia in cats.

Background and Purpose We tested the hypothesis that inhibition of nitric oxide synthase activity in brain before ischemia alters cerebral blood flow and decreases brain injury after 4 hours of middle cerebral artery occlusion in cats. Methods Halothane-anesthetized cats underwent 4 hours of left middle cerebral artery occlusion after they were randomly assigned to receive either intravenous Nω-nitro-L-arginine methyl ester, at a dose that completely inhibited cortical nitric oxide synthase activity (10 mg/kg, n=10), or an equal volume of diluent (10 mL saline, n=10). Serial blood flow measurements were made with radiolabeled microspheres, and injury volume was measured by triphenyltetrazolium staining. Results Blood flow to caudate nucleus and inferior temporal cortex decreased to the same extent in both groups during middle cerebral artery occlusion. Somatosensory evoked potential amplitude was reduced to less than 10% of baseline values in both groups. Injury volume of ipsilateral caudate nucleus in cats pretreated with nitroarginine (52 ± 5%, mean ± SE) was less (P < .05) compared with the saline group (80 ± 4%), whereas ipsilateral cerebral hemispheric injury volume was similar between the two groups (30 ± 6% and 32 ± 4% of hemisphere in saline and nitroarginine groups, respectively). Conclusions These results suggest that inhibition of nitric oxide synthase decreases caudate injury volume at 4 hours of middle cerebral artery occlusion without an alteration in distribution of cerebral blood flow. (Stroke. 1993;24:1717-1724.)

Treatment modalities for hypertensive patients with intracranial pathology: Options and risks

OBJECTIVES To review the cerebrovascular pathophysiology of hypertension, and the risks and benefits of antihypertensive therapies in the patient with intracranial ischemic or space-occupying pathology. DATA SOURCES Review of English language scientific and clinical literature, using MEDLINE search. STUDY SELECTION Pertinent literature is referenced, including clinical and laboratory investigations, to demonstrate principles of pathophysiology and controversies regarding the treatment of hypertension in patients with intracranial ischemic or space-occupying pathology. DATA EXTRACTION The literature was reviewed to summarize the pathophysiology, risks, and benefits of antihypertensive therapies in the patient with intracranial ischemic or space-occupying pathology. Treatment strategies were outlined with a particular emphasis on how antihypertensive agents may affect the brain. DATA SYNTHESIS Cerebral autoregulation typically occurs over a range of cerebral perfusion pressures between 50 and 150 mm Hg. Chronic hypertension results in adaptive changes that allow cerebral autoregulation to occur over a high range of pressures. Acute hypertension (rapid increase in perfusion pressure above the autoregulatory limit) may result in cerebral edema, persistent vasodilation, and brain injury. Treatment of a hypertensive emergency must be undertaken conservatively since the chronically hypertensive patient is at risk for ischemic brain injury when perfusion pressure is rapidly decreased beyond autoregulatory limits. In the patient with head injury or primary neurologic injury, acute antihypertensive intervention can result in further brain injury. Selection of appropriate antihypertensive therapy necessitates the careful consideration of agent-specific effects on cerebral blood flow, autoregulation, and intracranial pressure. For example, some vasodilators treat hypertension but also dilate the cerebral vasculature, and increase cerebral blood volume and intracranial pressure while decreasing cerebral perfusion pressure. Pharmacologic blockade of alpha 1- or beta 1-adrenergic receptors can reduce arterial blood pressure with little or no effect on intracranial pressure within the autoregulatory range. Like the direct peripheral vasodilators, calcium-channel antagonists are limited by cerebral vasodilation and increased intracranial pressure. Angiotensin converting enzyme inhibitors can also be used for mild to moderate hypertension but have the potential to further increase intracranial pressure in patients with intracranial hypertension. Barbiturates offer an alternative antihypertensive therapy since they decrease blood pressure as well as cerebral blood flow and oxygen metabolism. CONCLUSIONS The treatment of acute hypertension in the patient with intracranial ischemic or space-occupying pathology requires an understanding of the pathophysiology of hypertension and determinants of cerebral perfusion pressure. Individual agents should be selected based on their ability to promptly and reliably decrease blood pressure, while considering effects on cerebral blood flow and intracranial pressure.

Intravenous Basic Fibroblast Growth Factor Decreases Brain Injury Resulting From Focal Ischemia in Cats

BACKGROUND AND PURPOSE We tested the hypothesis that intravenous administration of basic fibroblast growth factor (bFGF) during 4 hours of permanent focal ischemia would affect acute brain injury. METHODS Halothane-anesthetized cats underwent left middle cerebral artery (MCA) occlusion for 4 hours. Control cats received diluent (n = 14). Experimental cats were treated with bFGF at a rate of 5 (n = 13), 50 (n = 13), or 250 microg/kg per hour (n = 9) intravenously beginning 60 minutes after initiation of ischemia and continuing until the end of the protocol. RESULTS As measured by the microsphere method, blood flow to ipsilateral caudate nucleus and ipsilateral inferior temporal cortex was decreased similarly during ischemia, before drug administration, in all groups. Likewise, there was no difference in blood flow to ipsilateral caudate nucleus or inferior temporal cortex as a result of bFGF administration during MCA occlusion. Triphenyltetrazolium-determined injury volume of the ipsilateral cerebral cortex (control, 40+/-7%; bFGF 5 microg/kg per hour, 22+/-5%; bFGF 50 microg/kg per hour, 26+/-7%; bFGF 255 microg/kg per hour, 23+/-6% of ipsilateral cerebral cortex; mean+/-SEM) was less in cats treated with bFGF. There was no difference among groups in injury volume to caudate nucleus (control, 29+/-8%; bFGF 5 microg/kg per hour, 29+/-8%; bFGF 50 microg/kg per hour, 21+/-7%; bFGF 250 microg/kg per hour, 32+/-7% of ipsilateral caudate nucleus). Somatosensory evoked potential amplitude decreased similarly (to <20% of baseline amplitude in all groups) during MCA occlusion and was not altered by bFGF administration. CONCLUSIONS; These data indicate that systemic administration of bFGF ameliorates acute injury in the cerebral cortex without increasing blood flow during focal ischemia in cats. Because bFGF afforded protection when administered after the onset of ischemia, bFGF may provide its beneficial effect by limiting progression of injury in ischemic border regions.

Neuroprotective Effect of σ1-Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl) Piperidine (PPBP) Is Linked to Reduced Neuronal Nitric Oxide Production

Background and Purpose —The potent σ1-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) provides neuroprotection in experimental stroke. We tested the hypothesis that PPBP attenuates striatal tissue damage after middle cerebral artery occlusion (MCAO) by a mechanism involving reduction of ischemia-evoked nitric oxide (NO) production. Furthermore, we determined whether the agent fails to protect ischemic brain when neuronal nitric oxide synthase (nNOS) is genetically deleted or pharmacologically inhibited (selective nNOS inhibitor, 7-nitroindazole [7-NI]). Methods —Halothane-anesthetized adult male Wistar rats were subjected to 2 hours of MCAO by the intraluminal filament occlusion technique. All physiological variables were controlled during the ischemic insult. In vivo striatal NO production was estimated via microdialysis by quantification of local, labeled citrulline recovery after labeled arginine infusion. In a second series of experiments, nNOS null mutants (nNOSKOs) and the genetically matched wild-type (WT) strain were treated with 90 minutes of MCAO. Brains were harvested at 22 hours of reperfusion for measurement of infarction volume by triphenyltetrazolium chloride histology. Results —PPBP attenuated infarction volume at 22 hours of reperfusion in cerebral cortex and striatum and markedly attenuated NO production in ischemic and nonischemic striatum during occlusion and early reperfusion. Treatment with 7-NI mimicked the effects of PPBP. In WT mice, infarction volume was robustly decreased by both PPBP and 7-NI, but the efficacy of PPBP was not altered by pharmacological nNOS inhibition in combined therapy. In contrast, PPBP did not decrease infarction volume in nNOSKO mice. Conclusions —These data suggest that the mechanism of neuroprotection of PPBP in vivo is through attenuation of nNOS activity and ischemia-evoked NO production. Neuroprotective effects of PPBP are lost when nNOS is not present or is inhibited; therefore, PPBP likely acts upstream from NO generation and its subsequent neurotoxicity.

The Effect of Needle Gauge and Lidocaine ph on Pain During Intradermal Injection

Local anesthetics can produce pain during skin infiltration.We designed a randomized, prospective trial to determine whether needle gauge and/or solution pH affect pain during the intradermal infiltration of lidocaine. After approval by our institutions human studies review board, 40 healthy adult volunteers gave their consent to participate in this study. All of the volunteers randomly received four intradermal injections. Each volunteer was blinded as to the content of the intradermal injections and to which needle size was used for each injection. Each volunteer randomly received a 0.25-mL intradermal injection of the following four solutions: 1) lidocaine 2% administered through a 25-gauge needle (lido-25); 2) lidocaine 2% mixed with sodium bicarbonate (4 mL of 2% lidocaine plus 1 mL of sodium bicarbonate, pH 7.26) administered through a 25-gauge needle (lido-bicarb-25); 3) lidocaine 2% administered through a 30-gauge needle (lido-30); and 4) lidocaine 2% mixed with sodium bicarbonate (4 mL of 2% lidocaine plus 1 mL of sodium bicarbonate) administered through a 30-gauge needle (lido-bicarb-30). In each patient, the injection site was in the same region for each of the four injections. The skin wheal was tested for appropriate anesthesia using a 19-gauge needle on the skin wheal. A visual analog pain score was recorded after each intradermal injection. The pain scores were significantly higher in the lido-25 (3.2 +/- 0.2) group than in the lido-30 (2.5 +/- 0.3), lido-bicarb-25 (1.9 +/- 0.2), and lido-bicarb-30 (1.3 +/- 0.2) groups. The lido-bicarb-30 injection was also rated as less painful than the lido-30 injection. We found no differences between the lido-bicarb-25 and the lido-bicarb-30 injections. Complete analgesia for the 19-gauge needle pain stimulus was achieved in all patients for each injection. We conclude that, overall, the pain intensity of an intradermal injection of 2% lidocaine is low. The addition of sodium bicarbonate to 2% lidocaine decreases the pain associated with an intradermal skin wheal, and although the use of a 30-gauge needle decreases the pain of injection, the addition of sodium bicarbonate seems to have a greater overall effect than needle size. Implications: Forty volunteers randomly received four intradermal injections consisting of 2% lidocaine with or without sodium bicarbonate via a 25- or 30-gauge needle. The addition of bicarbonate had a greater overall effect than needle size in decreasing the pain associated with the intradermal injection of lidocaine. (Anesth Analg 1998;86:379-81)

Correlation of the Average Water Diffusion Constant with Cerebral Blood Flow and Ischemic Damage After Transient Middle Cerebral Artery Occlusion in Cats

Magnetic resonance water diffusion imaging can detect early ischemic changes in stroke. Using a middle cerebral artery occlusion model, we examined which range of values of the orientation-independent diffusion quantity is an early noninvasive indicator of reduced cerebral perfusion and focal brain injury. Cats underwent either a 30-min occlusion followed by 3.5 h reperfusion (n = 7) or a 60-min occlusion followed by 4-h reperfusion (n = 6). Repeated measurements of CBF were made with radiolabeled microspheres, and acute focal injury was measured with triphenyltetrazolium chloride (TTC) staining. During occlusion, the decrease in Dav correlated with CBF for caudate [30-min occlusion (n = 13): p < 0.0001; 60-min occlusion (n = 6): p < 0.02] and for cortex [30-min occlusion (n = 12): p < 0.0001; 60-min occlusion (n = 5): p < 0.04]. Variable caudate and hemispheric injury levels were found among cats in both groups. The area of tissue injury demarcated by TTC began to correlate with the area of reduced Dav by 30 min of occlusion (p < 0.02), and this correlation improved (p < 0.0001) at 1, 1.5, and 2.0 h after the onset of occlusion. The time necessary to reach a one-to-one correspondence between the percent of hemisphere injured and the percent of hemispheric area with Dav < 0.65 × 10−9 m2/s was 2 h after occlusion. Thus, the absolute value of Dav is a good indicator of the risk of tissue injury, whereas the combination of Dav and the length of time of Dav reduction is an excellent predictor of acute focal tissue injury demarcated by TTC staining.

Critical care and perioperative management in traumatic spinal cord injury

&NA; Traumatic spinal cord injury is frequently associated with brain injury and with alterations in respiratory and cardiovascular function that require critical care management. Complications include respiratory failure, atelectasis, pneumonia, neurogenic shock, autonomic dysreflexia, venous thromboembolism, and sepsis. While complications may be managed with supportive care, the goal of ameliorating neurologic outcome has proved elusive. Methylprednisolone, when instituted <8 hours after traumatic spinal cord injury, was associated in two clinical trials with statistically significant improvements in motor scores at 6 months and 1 year; however, critical reappraisal of these data raises questions about their validity and clinical relevance. Until more evidence of clinically effective therapies is available, acute management must be driven by pathophysiologic principles, with emphasis on interventions that attenuate secondary neurologic injury; these include the rational use of immobilization, cautious airway management, and promotion of cord perfusion and oxygenation with the appropriate level of hemodynamic and respiratory support. Clinical trials of pharmacologic neuroprotection have yielded disappointing results, but the ongoing elucidation of spinal cord repair and regenerative mechanisms suggests new therapeutic prospects.

Monoclonal leukocyte antibody does not decrease the injury of transient focal cerebral ischemia in cats.

We tested the hypothesis that inhibition of leukocyte function by administration of monoclonal antibody 60.3 (MoAb 60.3) improves electrophysiological recovery and decreases injury volume following transient focal cerebral ischemia in cats. Methods Halothane-anesthetized cats underwent 90 minutes of left middle cerebral artery and bilateral common carotid artery occlusion followed by 180 minutes of reperfusion. Cats were assigned to receive either 2 mg/kg MoAb 60.3 (n=8) directed at the CDw18 leukocyte antigen complex or an equal volume of diluent (sterile saline; n=10) at 45 minutes of ischemia in a blinded fashion. Results Blood flow to the left temporoparietal cortex decreased to <5 ml/min/100 g with ischemia, but was minimally affected on the right side. Postischemic hyperemia occurred in the left caudate nucleus, whereas blood flow in other brain regions returned to control. No region demonstrated delayed hypoperfusion, and there were no differences between groups. Somatosensory evoked potential recorded over the left cortex was ablated during ischemia and recovered to <10% of baseline amplitude at 180 minutes of reperfusion in both groups. Left hemispheric injury volume, as assessed by 2,3,5 -triphenyltetrazolium chloride staining, was not affected by drug treatment (mean±SE values: MoAb 60.3, 37±5%; placebo, 38±7% of hemisphere). Conclusions Inhibition of leukocyte function with MoAb 60.3 does not afford protection from severe focal ischemia and reperfusion in cats.