Samidorphan, an opioid receptor antagonist, attenuates drug-induced increases in extracellular dopamine concentrations and drug self-administration in male Wistar rats.

Opioid receptors modulate neurochemical and behavioral responses to drugs of abuse in nonclinical models. Samidorphan (SAM) is a new molecular entity that binds with high affinity to human mu- (µ), kappa- (κ), and delta- (δ) opioid receptors and functions as a µ-opioid receptor antagonist with partial agonist activity at κ- and δ-opioid receptors. Based on its in vitro profile, we hypothesized that SAM would block key neurobiological effects of drugs of abuse. Therefore, we assessed the effects of SAM on ethanol-, oxycodone-, cocaine-, and amphetamine-induced increases in extracellular dopamine (DAext) in the nucleus accumbens shell (NAc-sh), and ethanol and cocaine self-administration behavior in rats. In microdialysis studies, administration of SAM alone did not result in measurable changes in NAc-sh DAext when given across a large range of doses. However, SAM markedly decreased average and maximal increases in NAc-sh DAext produced by each of the drugs of abuse tested. In behavioral studies, SAM attenuated fixed-ratio ethanol self-administration and progressive ratio cocaine self-administration. These results highlight the potential of SAM to counteract the neurobiological and behavioral effects of several drugs of abuse with differing mechanisms of action.

Opioid receptor modulation of neural circuits in depression: What can be learned from preclinical data?

Major depressive disorder (MDD) is a heterogeneous clinical syndrome involving distinct pathological processes. Core features of MDD include anhedonia, reduced motivation, increased anxiety, negative affective bias, cognitive impairments, and dysregulated neuroplasticity mechanisms. There are multiple biological hypotheses related to MDD, including dysfunction of the opioid system. Although opium was abandoned as an antidepressant after the introduction of monoaminergic drugs, there has been renewed interest in targeting the opioid system for MDD. In this review, we discuss the preclinical support of this idea using a neurocircuitry- and molecular neuroplasticity-based approach. This article highlights how the opioid system potently modulates mesolimbic circuitry underlying motivation and reward processing, limbic circuitry underlying fear and anxiety responses, cortical and hippocampal circuitry underlying a variety of cognitive functions, as well as broad functional and structural plasticity mechanisms. Ultimately, a more thorough understanding of how the opioid system modulates these core functional domains may lead to novel treatment strategies and molecular targets in the treatment of MDD.

Samidorphan mitigates olanzapine-induced weight gain and metabolic dysfunction in rats and non-human primates.

BACKGROUND: Olanzapine, regarded as one of the most efficacious antipsychotic medications for the treatment of schizophrenia, is associated with a high risk of weight gain and metabolic dysfunction. ALKS 3831, a clinical candidate for treatment of schizophrenia, is a combination of olanzapine and samidorphan, an opioid receptor antagonist. The addition of samidorphan is intended to mitigate weight gain and the metabolic dysregulation associated with the use of olanzapine. METHODS: Non-clinical studies were conducted to assess the metabolic effects of olanzapine and samidorphan alone and in combination at clinically relevant exposure levels. RESULTS: Chronic olanzapine administration in male and female rats shifted body composition by increasing adipose mass, which was accompanied by an increase in the rate of weight gain in female rats. Co-administration of samidorphan normalized body composition in both sexes and attenuated weight gain in female rats. In hyperinsulinemic euglycemic clamp experiments conducted prior to measurable changes in weight and/or body composition, olanzapine decreased hepatic insulin sensitivity and glucose uptake in muscle while increasing uptake in adipose tissue. Samidorphan appeared to normalize glucose utilization in both tissues, but did not restore hepatic insulin sensitivity. In subsequent studies, samidorphan normalized olanzapine-induced decreases in whole-body glucose clearance following bolus insulin administration. Results from experiments in female monkeys paralleled the effects in rats. CONCLUSIONS: Olanzapine administration increased weight gain and adiposity, both of which were attenuated by samidorphan. Furthermore, the combination of olanzapine and samidorphan prevented olanzapine-induced insulin insensitivity. Collectively, these data indicate that samidorphan mitigates several metabolic abnormalities associated with olanzapine in both the presence and the absence of weight gain.

Investigation of the reinforcing potential of samidorphan and naltrexone by fixed and progressive ratio intravenous self-administration testing in heroin-maintained rats.

BACKGROUND: Samidorphan is a novel µ-opioid antagonist with low intrinsic activity at κ- and δ-opioid receptors. AIMS: Because samidorphan is central nervous system-active, we investigated whether samidorphan (13.6, 40.8, 68 µg/kg/injection) served as a positive reinforcer in rats trained to self-administer heroin on a fixed ratio-5 schedule. Samidorphan's relative reinforcing effect was evaluated by progressive ratio/break-point determination. Naltrexone (13.6, 40.8, 68 µg/kg/injection) and heroin (7.5, 15, 25 µg/kg/injection) were comparators. RESULTS: All heroin doses maintained self-administration on fixed ratio-5 and progressive ratio/break-points at levels significantly greater than saline. Samidorphan and naltrexone had similar profiles on fixed ratio-5 with one samidorphan dose serving as a positive reinforcer and one naltrexone dose showing a strong trend ( p=0.053) for positive reinforcement. The numbers of injections of every samidorphan and naltrexone dose were significantly lower than all heroin doses. The numbers of self-administered samidorphan and naltrexone injections/session on fixed ratio-5 were not significantly different from one another. The mean inter-injection intervals for heroin were significantly shorter than for saline, whereas those of samidorphan and naltrexone were not. Progressive ratio break-points for samidorphan and naltrexone were not different from saline except for the highest dose of samidorphan. In addition, the progressive ratio break-points for samidorphan were not significantly different from those of naltrexone and were significantly lower than heroin. The samidorphan unit-doses evaluated in self-administration yielded plasma concentrations ranging between 25-109% and 10-45% of the maximum concentration values in humans. CONCLUSIONS: Overall, the profiles of samidorphan and naltrexone, which has no abuse liability, were similar in this model.

Opioid system modulators buprenorphine and samidorphan alter behavior and extracellular neurotransmitter concentrations in the Wistar Kyoto rat.

Approximately two-thirds of major depressive disorder (MDD) patients do not respond adequately to current therapies. BUP/SAM (ALKS 5461), a combination of buprenorphine (BUP) and samidorphan (SAM), is a novel opioid system modulator in development as an adjunct treatment for MDD. Using a rat strain (Wistar Kyoto rat) that is predisposed to stress and has an inadequate response to selective serotonin reuptake inhibitors (SSRIs), we investigated the effect of BUP and SAM, individually and in combination, in established nonclinical assays used to study antidepressants (the forced swim test, FST) and anxiolytics (marble burying test). As opioids and their receptors are expressed in mesocorticolimbic regions of the brain, we analyzed extracellular concentrations of dopamine, serotonin, and/or their metabolites in brain areas associated with mood and motivation. BUP alone and in combination with SAM significantly reduced immobility in the FST. Similarly, the BUP/SAM combination significantly reduced immobility in SSRI (escitalopram)-treated rats. BUP/SAM also decreased burying behavior. SAM attenuated BUP-induced changes of extracellular levels of serotonin and dopamine in the medial prefrontal cortex and nucleus accumbens shell. The latter suggests that the addition of SAM to BUP may limit activation of the mesolimbic dopamine reward pathway and thereby reduce BUP's reinforcing properties. SAM alone had no effect on neurochemistry or immobility in the FST. Collectively, these data indicate that opioid system modulation may offer an alternative mechanism that does not rely on enhanced serotonergic neurotransmission in neurocircuits associated with antidepressant and anxiolytic activity in nonclinical models.

Antidepressant-like effects of 3-carboxamido seco-nalmefene (3CS-nalmefene), a novel opioid receptor modulator, in a rat IFN-α-induced depression model.

Patients receiving the cytokine immunotherapy, interferon-alpha (IFN-α) frequently present with neuropsychiatric consequences and cognitive impairments. Patients (25-80%) report symptoms of depression, including, anhedonia, irritability, fatigue and impaired motivation. Our lab has previously demonstrated treatment (170,000IU/kg sc, 3 times per week for 4weeks) of the pro-inflammatory cytokine, IFN-α, induced a depressive phenotype in rats in the forced swim test (FST). Here, we examine the biological mechanisms underlying behavioral changes induced by IFN-α, which may be reflective of mechanisms underlying inflammation associated depression. We also investigate the potential of 3-carboxamido seco-nalmefene (3CS-nalmefene), a novel opioid modulator (antagonist at mu and partial agonist at kappa and delta opioid receptors in vitro), to reverse IFN-α induced changes. In vitro radioligand receptor binding assays and the [35S] GTPγS were performed to determine the affinity of 3CS-nalmefene for the mu, kappa and delta opioid receptors. IFN-α treatment increased circulating and central markers of inflammation and hypothalamic-pituitaryadrenal (HPA) axis activity (IL-6, IL-1ß and corticosterone) while increasing immobility in the FST, impairing of object displacement learning in the object exploration task (OET), and decreasing neuronal proliferation and brain-derived neurotrophic factor (BDNF) in the hippocampus. Treatment with 3CS-nalmefene (0.3mg/kg/sc twice per day, 3 times per week for 4weeks) prevented IFN-α-induced immobility in the FST and impaired object displacement learning. In addition, 3CS-nalmefene prevented IFN-α-induced increases in inflammation and hyperactivity of the HPA-axis, the IFN-α-induced reduction in both neuronal proliferation and BDNF expression in the hippocampus. Overall, these preclinical data would support the hypothesis that opioid receptor modulation is a relevant target for treatment of depression.

Prodrugs of pioglitazone for extended-release (XR) injectable formulations.

N-Acyloxymethyl derivatives of pioglitazone (PIO) have been prepared and characterized as model candidates for extended-release injectable formulations. All PIO derivatives prepared are crystalline solids as determined by powder X-ray diffraction, and the solubility in aqueous media is below 1 µM at 37 °C. The melting points steadily increase from 55 °C, for the hexanoyloxymethyl derivative, to 85 °C, for the palmitoyloxymethyl derivative; inversely, the solubilities in ethyl oleate decrease as a function of increasing acyl chain length. The butyroyloxymethyl ester has a higher melting point and a lower solubility in ethyl oleate than expected from the trend. The (13)C solid-state NMR spectra of the PIO homologues between the hexanoyloxymethyl derivative and stearoyloxymethyl derivative suggest a common structural motif with the acyl chains exchanging between two distinct conformations, and the rate of exchange is slower for longer chain derivatives. The butyroyloxymethyl derivative is efficiently converted to PIO in in vitro rat plasma with a half-life of <2 min at 37 (o) C, while the rate of enzymatic cleavage in rat plasma decreases as the ester chain length increases for the longer acyloxymethyl derivatives. The concentration of PIO in plasma increases rapidly, or "spikes," in the hours following intramuscular (IM) injection of either the HCl salt or the butyroyloxymethyl derivative. In contrast, the more lipophilic palmitoyloxymethyl derivative provides slow growth in the PIO concentration over the first day to reach levels that remain steady for 2 weeks. On the basis of its in vivo pharmacokinetic profile, as well as material and solubility properties, the PIO palmitoyloxymethyl derivative has potential as a once-monthly injectable medication to treat diabetes.

Effects of extended-release injectable naltrexone on self-injurious behavior in rhesus macaques (Macaca mulatta).

Self-injurious behavior (SIB) is a spontaneous behavior that threatens the health and wellbeing of multiple species. In humans, the opioid antagonist naltrexone hydrochloride has been used successfully to modulate the endogenous opioid system and reduce the occurrence of SIB. This study is the first to assess the efficacy of extended-release naltrexone in the pharmacologic treatment of SIB in rhesus macaques (Macaca mulatta). In an acute pharmacokinetic study of 4 macaques, we determined the mean naltrexone plasma concentration was maintained above the therapeutic level (2 ng/mL) after administration of a single dose (20 mg/kg) of 28-d extended-release naltrexone throughout the release period. For a subsequent treatment study, we selected 8 singly housed macaques known to engage in SIB. The study comprised a 4-wk baseline phase; an 8-wk treatment phase, during which each macaque received 2 doses of extended-release naltrexone 28 d apart; and a 4-wk posttreatment phase. Plasma samples were collected and analyzed weekly for naltrexone concentrations throughout the treatment and posttreatment phases. In addition, total of 6 h of video was analyzed per animal per phase of the study. Compared with baseline phases, both the frequency and the percentage of time spent displaying SIB decreased during the treatment phase, and the percentage of time remained decreased during the posttreatment phase. In contrast, extended-release naltrexone did not alter the expression of other abnormal, anxiety-related, or agonistic behaviors nor were levels of inactivity affected. The present study supports the use of naltrexone in the treatment of SIB in rhesus macaques.

Effects of oral loperamide on efficacy of naltrexone, baclofen and AM-251 in blocking ethanol self-administration in rats.

Naltrexone is a µ-opioid receptor antagonist that has been extensively studied for its ability to block the rewarding effects of ethanol. Opioid receptors are widely distributed within the gastrointestinal tract (GIT). Typically, naltrexone is administered by parenteral routes in nonclinical studies. We initially tested if opioid receptors within the GIT would influence the ability of oral naltrexone to inhibit ethanol oral self-administration in rats using the co-administration of oral loperamide, a peripherally restricted opioid agonist. As expected, oral naltrexone only had modest effects on ethanol intake, and the response was not dose-dependent. However in rats, treatment with loperamide prior to the administration of naltrexone resulted in a suppression of ethanol intake which approached that observed with naltrexone given by the subcutaneous (SC) route. Importantly, administration of loperamide prior to administration of naltrexone did not alter blood concentrations of naltrexone. We then evaluated if oral loperamide would enhance effects of baclofen (a GABA(B) receptor agonist) and AM-251 (a CB-1 receptor antagonist) and found that pre-treatment with loperamide did potentiate the action of both drugs to reduce ethanol self-administration. Finally, the specific opioid receptor type involved was investigated using selective µ- and κ-receptor antagonists to determine if these would affect the ability of the AM-251 and loperamide combination to block ethanol drinking behavior. The effect of loperamide was blocked by ALKS 37, a peripherally restricted µ-receptor antagonist. These data suggest an important role for opioid receptors within the GIT in modulating central reward pathways and may provide new insights into strategies for treating reward disorders, including drug dependency.

Naltrexone decreases D-amphetamine and ethanol self-administration in rhesus monkeys.

Amphetamines are the second most highly abused illicit drugs worldwide, yet there is no pharmacological treatment for amphetamine abuse and dependence. Preclinical studies and, more recently, human studies, suggest that the opioid receptor antagonist, naltrexone, might be useful in the treatment of amphetamine abuse. Naltrexone, an opioid receptor antagonist, is currently used for the treatment of alcohol dependence. The aim of this study was to explore the ability of naltrexone to modify self-administration of amphetamine or ethanol in rhesus monkeys. Monkeys were trained to respond to intravenous injections of either D-amphetamine (0.003 mg/kg/injection) or ethanol (0.05 g/kg/injection) on a fixed ratio 30 schedule. Naltrexone (0.01-1 mg/kg) was administered intramuscularly 30 min before the start of treatment test sessions. Naltrexone dose-dependently decreased both amphetamine and ethanol self-administration. These findings support the potential use of naltrexone as therapy for amphetamine and polydrug abuse.

Route of administration affects the ability of naltrexone to reduce amphetamine-potentiated brain stimulation reward in rats.

Opioid receptor antagonism has been shown to attenuate behavioral and neurochemical effects of amphetamine in humans and rodents. The effects of acute (oral or subcutaneous) or extended-release naltrexone (XR-NTX) were tested on the reward-enhancing effects of amphetamine using the intracranial self-stimulation (ICSS) paradigm. Acute exposure to drugs of abuse reduces the locus of rise (LOR) in the ICSS procedure, reflecting enhanced brain stimulation reward (BSR). Rats were treated once a day with naltrexone orally (PO; 5.0 mg/kg) or subcutaneously (SC; 0.5 mg/kg) for four consecutive days and tested with D-amphetamine (0.5 mg/kg, intraperitoneal) in the ICSS paradigm 30 minutes later on days 1 and 4. Separate groups of rats received XR-NTX (50 mg/kg, SC) or placebo microspheres (similar mass to XR-NTX, SC) on day 0 and tested with D-amphetamine in the ICSS paradigm on days 4, 14, 21, 28 and 41 after administration. Naltrexone plasma concentrations were determined for each amphetamine testing session using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). In rats pretreated with naltrexone acutely, amphetamine-potentiated BSR did not differ from vehicle-pretreated rats on either day 1 or day 4 (25-30% decrease in LOR). In XR-NTX-pretreated rats, amphetamine-potentiated BSR was reduced by 64 and 70% on days 4 and 14, respectively, compared to placebo microsphere-treated controls. This effect dissipated by day 21. Naltrexone plasma concentrations were comparable across all treatment groups (14-30 ng/ml) on days 1, 4 and 14. In summary, an extended-release formulation of naltrexone results in significant attenuation of psychostimulant-enhanced BSR that is not observed with acute naltrexone.

Pharmaceutical treatment for cognitive deficits in Alzheimer's disease and other neurodegenerative conditions: exploring new territory using traditional tools and established maps.

RATIONALE: Over 30 years ago, we began to develop a nonhuman primate model to study cognitive deficits of age-related neurodegenerative diseases and their neuroanatomical-neurochemical underpinnings for purposes of translating this work toward first pharmacotherapies. This effort produced several notable findings that eventually received consensus support, which we have been asked to review. OBJECTIVES: A discussion of these findings, in the context of issues and obstacles confronted and principles applied, might facilitate the development of even more effective models and treatments, not only for Alzheimer's disease (AD) but for many other disorders involving cognitive deficits. RESULTS: Collectively, our research provided first evidence of the following: aged primates can be used as 'models' for human age-related neurodegenerative diseases; key cognitive deficits in early AD share important conceptual similarities to deficits in both aged monkeys as well as non-demented humans (e.g., age-associated memory impairment and mild cognitive impairment); pharmacological intervention can reduce age-related cognitive impairments in animals that are conceptually similar to those seen in human diseases, including AD; cholinergics would likely be the first approved therapeutics for AD; and that many other classes of drugs would not likely succeed. CONCLUSIONS: Despite the early promise shown by behavioral/functional approaches to develop treatment strategies, the dramatic shift in focus away from behavioral outcomes in animal neurodegenerative research that began 20 years ago has compromised further progress and continues to impede our ability to understand how these diseases impair human cognition and what pathways might lead to effective therapies. Principles applied successfully in the past should provide guidance for facilitating efforts in the future.

Overriding the blockade of antinociceptive actions of opioids in rats treated with extended-release naltrexone.

A monthly extended-release formulation of the opioid antagonist naltrexone (XR-NTX) is approved for treatment of alcohol dependence. There is little research regarding overriding chronic (>21 days) competitive opioid receptor blockade with opioids for acute pain. Using the hot plate test after XR-NTX or placebo microsphere administration, rats were treated with an opioid analgesic to determine the dose required to produce the maximum response latency (MRL; 60 s). Rats were later treated with the same opioid to determine any potential effects on respiration rate or locomotor activity. In naïve rats, 15 mg/kg morphine, 0.1 mg/kg fentanyl and 8 mg/kg hydrocodone produced MRL. In XR-NTX treated rats, morphine produced 36% and 46% MRL at 90 mg/kg on days 4 and 19 and 96% MRL at 45 mg/kg on day 39. Fentanyl produced 100% MRL at 2.0 mg/kg on days 4 and 19 and at 0.5 mg/kg on day 39. Hydrocodone (80 mg/kg) produced 69%, 80% and 100% MRL on days 4, 19 and 39. Compared to placebo, these doses did not further depress respiration or alter locomotor activity. Thus, opioid receptor blockade with XR-NTX can be overcome in rats with higher doses of opioids without further affecting respiration or locomotor activity.

The preclinical development of Medisorb Naltrexone, a once a month long acting injection, for the treatment of alcohol dependence.

Oral naltrexone, a nonselective opioid antagonist, is approved for the treatment of alcohol and opioid dependence. However, the efficacy of oral naltrexone is limited by poor patient compliance. To overcome this limitation, attempts have been made to develop an injectable extended-release formulation of naltrexone, including encapsulation into biodegradable polymer microspheres (e.g. Medisorb Naltrexone, Vivitrex (naltrexone long acting injection)). In 1980, NIDA established development goals that they considered optimal for an extended-release formulation. At Alkermes, different formulations were tested with in vitro assays and in vivo models to select a lead formulation. Pharmacokinetic studies in rats confirmed that the principle formulation produced stable, pharmacologically relevant plasma levels of naltrexone for approximately one month following a single injection. The pharmacodynamic effects (antagonism of morphine analgesia) of extended-release naltrexone corresponded well with the pharmacokinetic profile from the same animals. While brain mu-opioid receptor density was found to increase over time in these rats, it did not appear to affect the ability of naltrexone to suppress morphine analgesia. Finally the pharmacokinetic profile of extended-release naltrexone in monkeys confirmed long duration of elevated plasma concentrations of naltrexone. Both naltrexone and the PLG polymer matrix in which it is encapsulated are well tolerated. Clinical trials of Vivitrex are currently ongoing in alcohol dependent patients.

Vivitrex, an injectable, extended-release formulation of naltrexone, provides pharmacokinetic and pharmacodynamic evidence of efficacy for 1 month in rats.

While oral naltrexone is effective in treating alcohol and opiate dependencies, poor patient adherence and widely fluctuating plasma levels limit its efficacy. To overcome these problems, an extended-release formulation of naltrexone (Vivitrex) was developed by encapsulating naltrexone into injectable, biodegradable polymer microspheres. Pharmacokinetic studies in rats demonstrated that this formulation produced stable, pharmacologically relevant plasma levels of naltrexone for approximately 1 month following either subcutaneous or intramuscular injections. While rats receiving placebo microspheres demonstrated a pronounced analgesic response to morphine in the hot-plate test, morphine analgesia was completely blocked in rats treated with extended-release naltrexone. This antagonism began on day 1 following administration and lasted for 28 days. Rats reinjected with extended-release naltrexone 34 days after the initial dose and tested for another 35 days showed consistent suppression of morphine analgesia for an additional 28 days. mu-Opioid receptor density, as measured by [(3)H]DAMGO autoradiography, increased up to two-fold following a single injection of extended-release naltrexone. Saturation binding assays using [(3)H]DAMGO showed changes in the midbrain and striatum at 1 week after extended-release naltrexone administration, and after 1 month in the neocortex. These receptor increases persisted for 2-4 weeks after dissipation of the morphine antagonist actions of naltrexone. These data suggest that therapeutically relevant plasma levels of naltrexone can be maintained using monthly injections of an extended-release microsphere formulation, and that changes in mu-opioid receptor density do not impact its efficacy in suppressing morphine-induced analgesia in the rat. Clinical trials of extended release naltrexone for treating alcohol and opiate dependency are currently ongoing.

Sustained release chemotherapeutic microspheres provide superior efficacy over systemic therapy and local bolus infusions.

PURPOSE: The present studies evaluated the ability of injectable, biodegradable microspheres releasing carboplatin, doxorubicin, or 5-fluorouracil to suppress the growth of solid tumors implanted subcutaneously or intramuscularly. METHODS: Seven to 10 days after implantation of MATB-III cells, rats received systemic chemotherapy, intratumoral bolus chemotherapy, or injections of chemotherapeutic microspheres into the tumor center or multiple sites along the outer perimeter of the tumor. RESULTS: A single treatment with carboplatin, doxorubicin, or 5-fluorouracil microspheres along the perimeter of the tumors produced a significant, dose-related suppression in tumor growth, relative to injections directly into the tumor center. Moreover, five temporally-spaced microsphere treatments along the tumor perimeter (with either doxorubicin or 5-fluorouracil microspheres) completely eradicated 100% of the subcutaneous tumors and 40-53% of the intramuscular tumors. Polypharmacy, accomplished by blending doxorubicin- and 5-fluorouracil-loaded microspheres and injecting them into the tumors was even more efficacious than sustained delivery of either drug alone. Comparable doses of systemic chemotherapy or intratumoral bolus chemotherapy were ineffective. CONCLUSIONS: Injectable microspheres might be ideal for local, sustained delivery of chemotherapeutic agents to solid tumors. However, attention must be paid to the placement of the microspheres, for injections around the tumor perimeter may be required for efficacy.

The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct?

Data accumulated over the last 10 years have led to the popular hypothesis that neutrophils and other inflammatory cells play a prominent role in the neuropathology of cerebral ischemia. This hypothesis was derived from a large number of studies involving three general observations: (1) leukocytes, particularly neutrophils, are present in ischemic tissue at the approximate time that substantial neuronal death occurs; (2) neutropenia is sometimes associated with reduced ischemic damage; and (3) treatments that prevent leukocyte vascular adhesion and extravasation into the brain parenchyma can be neuroprotective. This review reexamines the literature to ascertain its support for a pathogenic role for neutrophils in ischemia-induced neuronal loss. To accomplish this goal, we employed several logical theorems of "cause-effect" relationships, as they pertain to leukocytes and ischemic brain damage. Since the majority of studies focused on neutrophils as the most likely pathogenic inflammatory cell, this review necessarily does so here. We reasoned that if neutrophils play an important pathogenic (i.e., cause-effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia. This review of the literature reveals that the existing evidence does not readily support any of these predictions and that, therefore, it consistently falls short of establishing a clear cause-effect relationship between leukocyte recruitment and the pathogenesis of ischemia. While the available evidence does not necessarily rule out a potential pathogenic role of neutrophils and other leukocytes, it nevertheless does expose serious weaknesses in the existing studies intended to support that hypothesis. For this reason we also offer suggestions for additional experiments and the inclusion of control groups that, in the future, might provide more effective or conclusive tests of the hypothesis.