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Background Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are increased in the brain after experimental ischemic stroke in rats. These two proteases are involved with the degradation of the basal lamina and loss of stability of the blood brain barrier that occurs after ischemia and that is associated with thrombolytic therapy in ischemic stroke. Minocycline is a lipophilic tetracycline and is neuroprotective in several models of brain injury. Minocycline inhibits inflammation, apoptosis and extracellular matrix degradation. In this study we investigated whether delayed minocycline inhibits brain MMPs activated by ischemia in a model of temporary occlusion in Wistar rats. Results Both MMP-2 and MMP-9 were elevated in the ischemic tissue as compared to the contra-lateral hemisphere after 3 hours occlusion and 21 hours survival (p < 0.0001 for MMP-9). Intraperitoneal minocycline at 45 mg/kg concentration twice a day (first dose immediately after the onset of reperfusion) significantly reduced gelatinolytic activity of ischemia-elevated MMP-2 and MMP-9 (p < 0.0003). Treatment also reduced protein concentration of both enzymes (p < 0.038 for MMP-9 and p < 0.018 for MMP-2). In vitro incubation of minocycline in concentrations as low as 0.1 μg/ml with recombinant MMP-2 and MMP-9 impaired enzymatic activity and MMP-9 was more sensitive at lower minocycline concentrations (p < 0.05). Conclusion Minocycline inhibits enzymatic activity of gelatin proteases activated by ischemia after experimental stroke and is likely to be selective for MMP-9 at low doses. Minocycline is a potential new therapeutic agent to acute treatment of ischemic stroke.

The choroid plexuses (CPs) play pivotal roles in the most basic aspects of neural function. Some of the roles of the CP include maintaining the extracellular milieu of the brain by actively modulating chemical exchange between the CSF and brain parenchyma, surveying the chemical and immunological status of the brain, detoxifying the brain, secreting a nutritive “cocktail” of polypeptides, and participating in repair processes following trauma. This diversity of functions suggests that even modest changes in the CP can have far reaching effects. Indeed, changes in the anatomy and physiology of the CP have been linked to several CNS diseases. It is also possible that replacing diseased CP or transplanting healthy CP might be useful for treating acute and chronic brain diseases. Here we describe the wide-ranging functions of the CP, alterations of these functions in aging and neurodegeneration, and recent demonstrations of the therapeutic potential of transplanted CP for neural trauma.

The mechanisms underlying age-related loss of muscle and bone tissue are poorly understood but are thought to involve changes in sex hormone status, physical activity, and circulating levels of inflammatory cytokines. This study attempts to develop an animal model useful for evaluating these mechanisms in vivo. Male C57BL/6 mice were included for study at 3, 6, 12, 18, 24, and 29 months of age. Endocortical mineralizing surface, serum leptin, body weight, and percentage of body fat all increased between 6 and 12 months of age as activity level declined. Serum levels of the inflammatory marker IL-6 increased significantly after 12 months of age, following the observed increase in body weight and percent body fat. Hindlimb muscle mass declined significantly between 18 and 24 months of age, both absolutely and relative to total body mass, with a further decline ( approximately 15%) between 24 and 29 months. Loss of muscle mass after 18 months of age was accompanied by a significant increase in bone resorption, as indicated by serum pyridinoline cross-links, and a significant decrease in fat mass, serum leptin, bone strength, bone mineral density, and vertical cage activity. No significant changes in serum testosterone with aging were detected in the mice, as levels were essentially constant between 6 and 29 months. Our data show that mice lose a significant amount of muscle and bone tissue with age, and this loss of musculoskeletal tissue is accompanied by a drop in serum leptin and preceded by a significant decrease in physical activity.

Numerous reports elucidate that tissue-specific stem cells are phenotypically plastic and their differentiation pathways are not strictly delineated. Although the identity of all the epigenetic factors which may trigger stem cells to make a lineage selection are still unknown, the plasticity of adult stem cells opens new approaches for their application in the treatment of various disorders. There is increasing researcher interest in hematopoietic stem cells for treatment of not only blood-related diseases but also various unrelated disorders including neurodegenerative diseases. Human umbilical cord blood (hUCB) cells, due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, including neural cells, may be useful as an alternative cell source for cell-based therapies requiring either the replacement of individual cell types and/or substitution of missing substances. Here we focus on recent findings showing the robustness of adult stem cells derived from hUCB and their potential as a source of transplant cells for the treatment of diseased or injured brains and spinal cords. Depending upon the pathological microenvironment in which the hUCB cells are introduced, neuroprotective and/or trophic effects of these cells, from release of various growth or anti-inflammatory factors to moderation of immune-inflammatory effectors, may be more likely than neural replacement. These protective effects may prove essential to maintaining restored tissue integrity over the course of various diseases or injuries.

Neural transplantation is emerging as a viable long-term method to treat degenerative diseases of the nervous system. Unfortunately, the shortage of suitable donor tissue necessitates the development of novel approaches to enable transplantation on a widespread basis. Transplantable cells obtained from xenogeneic sources have shown considerable promise in preclinical animal models, as well as in a limited number of human trials conducted to date. Although animal-sourced tissue overcomes the shortage of available cells, transplant survival remains poor, and immunosuppressive regimens are suboptimal in improving graft viability, with deleterious side effects. One potential way of enabling xenotransplantation takes advantage of the immunoprotective capabilities of Sertoli cells, which are normally found in the testes, where they provide local immunologic protection to developing germ cells. In animal models, allogeneic and xenogeneic Sertoli cells survive and function for extended periods of time when grafted into the brain. Moreover, isolated Sertoli cells protect cografted dopaminergic neurons from immune destruction and reverse lesion-induced deficits in Parkinsonian rodents. This chapter discusses these benefits within the context of several potential underlying biological mechanisms.

Choroid plexus from neonatal pigs was encapsulated in alginate microcapsules and transplanted into the rat striatum. Three days later, the same animals received unilateral injections of quinolinic acid (225 nmol) into the ipsilateral striatum. Choroid plexus transplants ameliorated the weight loss and motor impairments resulting from QA. Histological analysis demonstrated that choroid plexus transplants reduced the volume of striatal damage and protected ChAT-, but not NADPH-diaphorase-positive neurons. These data are the first to demonstrate that transplanted choroid plexus cells can protect striatal neurons from excitotoxic damage and that this strategy may ultimately prove relevant for the treatment of Huntington's disease.

Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These results taken together suggest that DADLE stands as a novel therapeutic agent. In this review paper, we present laboratory evidence supporting the use of DADLE for protection of peripheral and central nervous system.

The choroid plexus (CP) produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. However, the CP may have additional functions in the CNS beyond these traditional roles. Preclinical and clinical studies in ageing and neurodegeneration demonstrate anatomical and physiological changes in CP, suggesting roles in normal and pathological conditions and potentially endogenous repair processes following trauma. One of the broadest functions of the CP is establishing and maintaining the extracellular milieu throughout the brain and spinal cord, in part by secreting numerous growth factors into the CSF. The endogenous secretion of growth factors raises the possibility that transplantable CP might enable delivery of these molecules to the brain, while avoiding the conventional molecular and genetic alterations associated with modifying cells to secrete selected products. This review describes some of the anatomical and functional changes of CP in ageing and neurodegeneration, and recent demonstrations of the therapeutic potential of transplanted CP for neural trauma.

We investigated the neuroprotective effects of immunosuppressant cyclosporine-A (CsA) and the anti-inflammatory methylprednisolone (MP) in a stroke model. Adult Sprague-Dawley rats underwent middle cerebral artery (MCA) occlusion then were randomly treated with either: low dose CsA, MP, low dose CsA plus MP, high dose CsA, or vehicle. Ischemic animals that received low dose CsA, MP or vehicle displayed profound motor and neurological impairments at days 1-3 after stroke. In contrast, ischemic animals that received high dose CsA exhibited near normal motor and neurological functions throughout the test period. Of note, ischemic animals that received low dose CsA plus MP showed significantly less motor and neurological deficits at day 1, but thereafter displayed behavioral impairments. Histological analysis at 3 days post-stroke revealed that only those ischemic animals treated with high dose CsA had significantly reduced cerebral infarcts. This study is the first report to demonstrate partial and transient neuroprotection against stroke by low dose CsA when combined with MP.

The present study examined the neuroprotective effects of immunosuppressant cyclosporine-A (CsA) and anti-inflammatory methylprednisolone (MP) in a stroke model. Adult Sprague-Dawley rats were initially subjected to transient middle cerebral artery occlusion (MCAo) then randomly assigned to one of the following treatment conditions: low dose CsA, MP, low dose CsA plus MP, high dose CsA, or vehicle. Ischemic animals that received low dose CsA, MP or vehicle exhibited significant cognitive impairments, as revealed by passive avoidance and Morris water maze tasks, at days 1-3 after stroke. In contrast, ischemic animals that received high dose CsA exhibited near normal cognitive performance throughout the test period. Ischemic animals that received low dose CsA plus MP also showed significantly less cognitive deficits but such attenuation of stroke-induced behavioral impairments was only consistently reflected in the passive avoidance task, while performance in the Morris water maze task deteriorated over time. Histological analysis at 3 days post-stroke revealed that only those ischemic animals treated with high dose CsA had significantly reduced cerebral infarcts. These observations suggest that despite overt cerebral damage, alterations in simple, but not complex, cognitive tasks produced by MCAo could be ameliorated by low dose CsA when combined with MP.

Methanesulfonyl fluoride (MSF), a highly selective CNS inhibitor of acetylcholinesterase, has been recently demonstrated to promote improvement in cognitive performance in patients with senile dementia of Alzheimer type. Because a similar cognitive impairment may accompany stroke, we investigated in the present study whether treatment with MSF could produce beneficial effects in adult rats subjected to an experimental stroke model. Sprague-Dawley rats received transient 60 min intraluminal occlusion of the right middle cerebral artery (MCAo) and were given i.p. injections of either MSF (1 mg/kg at 24 and 48 h post-MCAo and 0.3 mg/kg thereafter every other day) or the vehicle, peanut oil, for 4 weeks. Behavioral tests and biochemical assays were performed at 28 days post-surgery. MSF treatment produced about 90% inhibition of acetylcholinesterase in the brain. Ischemic animals that received the vehicle displayed significant elevated body swing biased activity (84.8 +/- 10%) and significantly prolonged acquisition (398 +/- 62 s) and shortened retention (79 +/- 26 s) of the passive avoidance task. Interestingly, while the ischemic animals that received the MSF exhibited elevated body swing biased activity (87.7 +/- 8%), they performed significantly better in the passive avoidance task (255 +/- 36 s and 145 +/- 18 s in acquisition and retention) than the vehicle-treated animals. Moreover, whereas brains from both groups of animals revealed similar extent and degree of cerebral infarction, the MSF-treated ischemic animals showed more intense immunoreactivity, as well as a significantly higher number (10-15% increase) of septal choline acetyltransferase-positive cells than the vehicle-treated ischemic animals. These results show that MSF, possibly by preserving a functional cholinergic system, attenuated stroke-induced deficits in a simple learning and memory task.

We examined in the present study alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler revealed that transplanted animals exhibited near normal cerebral blood flow (CBF, 150 perfusion units) at a much earlier period post-transplantation (day 4) compared to animals that received saline infusion (day 12) (p's

We monitored alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following middle cerebral artery occlusion (MCAo) and intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler and Evans Blue assay revealed that BMSC grafts dose-dependently restored CBF and BBB to near normal levels at a much earlier period (Days 4-5 post-MCAo) in transplanted stroke animals compared to stroke animals that received saline infusion (Days 11-14 post-MCAo). Xenografted BMSCs survived in the absence of immunosuppression, and elevated levels of transforming growth factor-beta superfamily of neurotrophic factors were detected in transplanted stroke animals. These data suggest that early restoration of CBF and BBB following transplantation of BMSCs could mediate the reported functional outcomes in stroke animals.

Chronic systemic melatonin treatment attenuates abnormalities produced by occlusion of middle cerebral artery (MCA) in adult rats. Because the pineal gland secretes high levels of melatonin, we examined in the present study whether transplantation of pineal gland exerted similar protective effects in MCA-occluded adult rats. Animals underwent same-day MCA occlusion and either intrastriatal transplantation of pineal gland (harvested from 2-month-old rats) or vehicle infusion. Behavioral tests (from day of surgery to 3 days posttransplantation) revealed that transplanted stroke rats displayed significantly less motor asymmetrical behaviors than vehicle-infused stroke rats. Histological analysis at 3 days posttransplantation revealed that transplanted stroke rats had significantly smaller cerebral infarction than vehicle-infused rats. Additional experiments showed that pinealectomy affected transplantation outcome, in that transplantation of pineal gland only protected against stroke-induced deficits in stroke animals with intact pineal gland, but not in pinealectomized stroke rats. Interestingly, nonpinealectomized vehicle-infused stroke rats, as well as pinealectomized transplanted stroke rats, had significantly lower melatonin levels in the cerebrospinal fluid than nonpinealectomized transplanted stroke rats. We conclude that intracerebral transplantation of pineal gland, in the presence of host intact pineal gland, protected against stroke, possibly through secretion of melatonin.

Part I: Immunosuppressants, Neurologic Disorders, and Neuroprotection Introduction: Immunosuppressants as Neuroprotective Agents Marcus F. Keep, Hiroyuki Uchino, and Eskil Elmer Part II: Immunosuppressants and Parkinson's Disease Cyclosporin-Mediated Amelioration of Degeneration of Dopaminergic Neurons in Experimental Models of Parkinsonism Norio Ogawa and Ken-ichi Tanaka Immunophilin Ligands and Dopamine Neurons: Specific Effects in Culture and In Vivo Lauren C. Costantini and Ole Isacson Effects of Neuroimmunophilin Ligands on Parkinson's Disease and Cognition Joseph P. Steiner, Douglas T. Ross, Hansjorg Sauer, Theresa Morrow, and Gregory S. Hamilton Improved Survival of Grafted Dopamine Neurons by Calcineurin Inhibitors Roger F. Castilho, Oskar Hansson, and Patrik Brundin Possible Mechanisms Underlying the Protective Action of Immunosuppressants Against Parkinson's Disease: The Mitochondrial Permeability Transition Pore Hypothesis L. V. P. Korlipara and A. H. V. Schapira Part III: Immunosuppressants and Other Age-Related Disorders Immunosuppressants and Alzheimer's Disease Mark P. Mattson Cyclosporin A Protects Striatal Neurons from Mitochondrial Dysfunction: Implications for Huntington's Disease Liza Leventhal and Jeffrey H. Kordower Part IV: Immunosuppressants, Stroke, and Traumatic Brain Injury Cyclosporin A Protects Mitochondria in an In Vitro Model of Hypoxia/Reperfusion Injury Vladimir Gogvadze and Christoph Richter Protective Effect of Cyclosporin A on Glial Activation and White Matter Alterations Induced by Chronic Cerebral Hypoperfusion Hideaki Wakita, Hidekazu Tomimoto, and Ichiro Akiguchi Blockade of Late-Onset Reduction of Muscarinic Acetylcholine Receptors by Immunosuppressants in Forebrain Ischemia Ken-ichi Tanaka, M. Asanuma, and NorioOgawa The Role of Immunophilins in Focal Cerebral Ischemia: Evidence of Neuroprotection by FK506 A. L. McGregor, P. A. Jones, J. F. McCarter, T. E. Allsopp, and J. Sharkey Immunosuppressants in Traumatic Brain Injury David O. Okonkwo and John T. Povlishock Part V: immunosuppressants and Spinal Cord Injury Inhibition of Lipid Peroxidation by Cyclosporin After Spinal Cord Injury in Rats Antonio Ibarra and Araceli Diaz-Ruiz Axonal Regeneration in Cyclosporin A-Treated Rats Submitted to Transverse Section of the Spinal Cord Guido Palladini and Brunella Caronti Part VI: Immunosuppressants and Sciatic Nerve Injury Neuroimmunophilin Ligands Accelerate and Promote Nerve Regeneration in the Rat Peripheral Nerve and Spinal Cord: Role of the Steroid Receptor-FKBP52 Complex Bruce G. Gold Neuroimmunophilin Ligands Stimulate Recovery of Injured Sciatic Nerves Joseph P. Steiner, Heather Valentine, Theresa Morrow, and Gregory Hamilton Part VII: Immunosuppressants and Other Disorders of the Central Nervous System Cyclosporin A Prolongs Survival of SOD1 Mutant Mice and Implicates Mitochondrial Permeability Transition in ALS Marcus F. Keep, Keith S. K. Fong, Katalin Csiszar, and Eskil Elmer Psychological Effects of Cyclosporine-A Shigeru Watanabe Index

Neural transplantation has emerged as an efficacious experimental treatment for CNS disorders, especially Parkinson's disease. However, logistical and ethical issues impede large-scale clinical trials. To this end, alternatives to human fetal cells as donor cell grafts have been examined, including xenografts, stem cells, genetically engineered cells, immortalized cell lines, or paraneural cells that secrete specific neurotrophic or growth factors. Accumulating evidence also suggests that exogenous treatment with neurotrophic or growth factors, immunosuppressants, free radical scavengers, and anti-apoptotic agents can enhance survival and functional effects of the grafts. This article will review recent studies demonstrating the potential of these alternative cell graft sources and novel drugs for treating Parkinson's disease.

Fetal rat kidney cells produce high levels of glial-derived neurotrophic factor (GDNF) and exert neuroprotective effects when transplanted into the brain in animal models of Parkinson's disease and stroke. The purpose of the present experiment was to produce kidney cell lines that secrete GDNF. Genes encoding two truncated N-terminal fragments of SV40 large T antigen, T155g and T155c, which does not code for small t antigen, were used. T155g was transduced into E17 cultured fetal Sprague-Dawley rat kidney cortex cells using a plasmid vector, and T155c was transduced with a plasmid and a retroviral vector. Sixteen clones were isolated from cultures transfected with the T155g-expressing plasmid. No cell lines were obtained with T155c. Four clones produced GDNF at physiological concentrations ranging from 55 to 93 pg/ml of medium. These four clones were transplanted into the ischemic core or penumbra of rats that had undergone middle cerebral artery occlusion (MCAO). Three of the four clones reduced the volume of infarction and the behavioral abnormalities normally resulting from MCAO. Blocking experiments with antibodies to GDNF and platelet-derived growth factor (PDGF) suggested that these growth factors contributed only minimally to the reduction in infarct volume and behavioral abnormality. These cell lines may be useful for intracerebral transplantation in animal models of brain injury, stroke, or Parkinson's disease.

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFβ1 and TGFβ2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

The role of glial cells in neuronal death has become a major research interest. Glial cell activation has been demonstrated to accompany cerebral ischemia. However, there is disagreement whether such gliosis is a cell death or a neuroprotective response. In the present study, we examined alterations in glial cell responses to the reported neuroprotective action of the free radical scavenger, melatonin, against cerebral ischemia. Adult male Wistar rats were given oral injections of either melatonin (26 micromol/rat) or saline just prior to 1 h occlusion of the middle cerebral artery (MCA), then once daily for 11 or 19 consecutive days. At 11 and 19 days after reperfusion of the MCA, randomly selected animals were killed and their brains removed for immunohistochemical assays. Melatonin significantly enhanced survival of glial cells (as revealed by glial cell specific markers, glial fibrillary acidic protein and aquaporin-4 immunostaining) at both time periods postischemia, and the preservation of these glial cells in the ischemic penumbra corresponded with a markedly reduced area of infarction (detected by immunoglobulin G and hematoxylin-eosin staining), as well as increased neuronal survival. The ischemia-induced locomotor deficits were partially ameliorated in melatonin-treated animals. In vitro replications of ischemia by serum deprivation or by exposure to free radical-producing toxins (sodium nitroprusside and 3-nitropropionic acid) revealed that melatonin (10 microg/ml or 100 microM) treatment of pure astrocytic cultures significantly reduced astrocytic cell death. These results suggest a potential strategy directed at enhancing glial cell survival as an alternative protective approach against ischemic damage.

A major problem in neural transplantation therapy is poor survival of grafted cells, which may be due to low cell viability prior to transplantation or scarce trophic factors available to the cells following transplantation. Recently, the delta enkephalin analogue [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) has been demonstrated to protect against, as well as to reverse methamphetamine-induced loss of dopamine transporters. Here, we show that pretreatment with DADLE (0.0025, 0.005, 0.01 g/ml) dose-dependently enhanced cell viability of cultured primary rat fetal mesencephalic cells. In addition, DADLE administration in adult rats (4 mg/kg every 2 h, 4 injections, i.p.) prior to 6-hydroxydopamine lesions of the medial forebrain bundle, significantly reduced the severity of loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra 1 month post-lesion. This is the first report suggesting that DADLE can be used as a supplement factor for improving the cell viability of fetal mesencephalic cells and as a protective agent against neurotoxicity in a Parkinson's disease model.

There are three main mechanisms of neuronal cell death which may act separately or cooperatively to cause neurodegeneration. This lethal triplet of metabolic compromise, excitotoxicity, and oxidative stress causes neuronal cell death that is both necrotic and apoptotic in nature. Aspects of each of these three mechanisms are believed to play a role in the neurodegeneration that occurs in both Parkinson's and Huntington's diseases. Strategies to rescue or protect injured neurons usually involve promoting neuronal growth and function or interfering with neurotoxic processes. Considerable research has been done on testing a large array of neuroprotective agents using animal models which mimic these disorders. Some of these approaches have progressed to the clinical arena. Here, we review neuroprotective strategies which have been found to successfully ameliorate the neurodegeneration associated with Parkinson's and Huntington's diseases. First, we will give an overview of the mechanisms of cell death and the background of Parkinson's and Huntington's diseases. Then we will elaborate on a range of neuroprotective strategies, including neurotrophic factors, anti-excitotoxins, antioxidants, bioenergetic supplements, anti-apoptotics, immunosuppressants, and cell transplantation techniques. Most of these approaches hold promise as potential therapies in the treatment of these disorders.

Glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1, also known as GDNFR-alpha) is a glycolipid-anchored membrane protein of the GFRalpha family, which binds glial cell line-derived neurotrophic factor [Jing S. et al. (1996) Cell 85, 1113-1124; Treanor J. J. et al. (1996) Nature 382, 80-83], a survival factor for several populations of central and peripheral neurons, including midbrain dopamine neurons [Lin L. F. et al. (1993) Science 260, 1130-1132], and mediates its ligand-induced cell response via a tyrosine kinase receptor called Ret [Takahashi M. et al. (1988) Oncogene 3, 571-578; Takahashi M. and Cooper G. M. (1987) Molec. Cell Biol. 7, 1378-1385]. In this paper, we show that mice with a null mutation of the GFRalpha1 gene manifest epithelial-mesenchymal interaction deficits in kidney and severe disturbances of intestinal tract development similar to those seen with glial cell line-derived neurotrophic factor or Ret null mutations. There is a marked renal dysgenesis or agenesis and the intrinsic enteric nervous system fails completely to develop. We also show that newborn GFRalpha1-deficient mice display no or minimal changes in dorsal root and sympathetic ganglia. This is in contrast to the deficits reported in these neuronal populations in glial cell line-derived neurotrophic factor and Ret null mutations. Mesencephalic dopaminergic neurons in the substantia nigra and ventral tegmental area appear intact at the time of birth of the mutated mice. Mice homozygous for the GFRalpha1 null mutation die within 24 h of birth because of uremia. Heterozygous animals, however, live to adulthood. There is a significantly reduced neuroprotective effect of glial cell line-derived neurotrophic factor in such heterozygous animals, compared with wild-type littermates, after cerebral ischemia. Taken together with previous data on glial cell line-derived neurotrophic factor and Ret, our results strongly suggest that GFRalpha1 is the essential GFRalpha receptor for signaling in the glial cell line-derived neurotrophic factor-Ret pathway in the kidney and enteric nervous system development, and that GFRalpha2 or GFRalpha3 cannot substitute for the absence of GFRalpha1. Moreover, neuroprotective actions of exogenous glial cell line-derived neurotrophic factor also require full GFRalpha1 receptor expression.

Part I. Mitochondrial Toxins: Symptomatology, Origin, and Chemistry. Clinical Manifestations and Mechanisms of Action of Environmental Mitochondrial Toxins, Mohammad I. Sabri, Peter S. Spencer, Safia Baggia, and Albert C. Ludolph. History of 3-Nitropropionic Acid: Occurrence and Role in Human and Animal Disease, Bradley F. Hamilton, Daniel H. Gould, and David L. Gustine. The Neurochemistry of 3-Nitropropionic Acid, Norman C. Reynolds, Jr. and Wen Lin. Part II. Mitochondrial Dysfunctions: Models of Neurodegeneration and Mechanisms of Action. In Vitro Studies of 3-Nitropropionic Acid, Gail D. Zeevalk. Cognitive and Motor Deficits Produced by Acute and Chronic Administration of 3-Nitropropionic Acid in Rats, Gary L. Dunbar, Deborah A. Shear, Jie Dong, and Kristi L. Haik-Creguer. Comparative Study on 3-Nitropropionic Acid Neurotoxicity, Cesario V. Borlongan. Mechanisms of 3-Nitropropionic Acid Neurotoxicity, James W. Geddes, Vimala Bondada, and Zhen Pang. Gender-Related Difference of the Effect of 3-Nitropropionic Acid on Striatal Artery, Keiya Nakajima, Yasunobu Shimano, Kunio Torii, and Hitoo Nishino. Variable Susceptibility to Neurotoxicity of Systemic 3-Nitropropionic Acid, Tajrena Alexi, Richard L. M. Faull, and Paul E. Hughes. The 3-Nitropropionic Acid Model of Huntington's Disease: Do Alterations in the Expression of Metabolic mRNAs Predict the Development of Striatal Pathology? Keith J. Page, Alicia Meldrum, and Stephen B. Dunnett. Mechanisms of Action of 3-Nitropropionic Acid: Dopamine Overflow and Vulnerability of the Lateral Striatal Artery, Michiko Kumazaki, Chucharin Ungsuparkorn, Shripad B. Deshpande, Atsuo Fukuda, and Hitoo Nishino. Mitochondrial Inhibition and Neuronal Death in Huntington's Disease, Maria Isabel Behrens. Effects of Brain Mitochondrial Metabolism, Aging, and Caloric Restriction on Membrance Lipids and Proteins: An Electron Paramagnetic Resonance Investigation, S. Prasad Gabbita, John M. Carney, and D. Allan Butterfield. Malonate: Profileand Mechanisms of Striatal Toxicity, Alicia Meldrum, Keith J. Page, Barry J. Everitt, and Stephen B. Dunnett. Malonic Acid and the Chronic Administration Model of Excitotoxicity, Terence J. Bazzett, Roger L. Albin, and Jill B. Becker. Sodium Azide-Induced Neurotoxicity, Yun Wang and Cesario V. Borlongan. Part III. Treatment Interventions for Mitochondrial-Induced Neurotoxicity. Neuroprotective Strategies Against Cellular Hypoxia, Matthias W. Riepe. Neuroprotective Effect of Perinatal Hypoxia Against 3-Nitropropionic Acid Neurotoxicity, Zbigniew K. Binienda and Andrew C. Scallet. Neural Transplantation and Huntington's Disease: What Can We Learn from the 3-Nitropropionic Acid Model? Cesario V. Borlongan, Christine E. Stahl, Thomas B. Freeman, Robert A. Hauser, and Paul R. Sanberg. Neuroprotective Strategies in Parkinson's Disease and Huntington's Chorea: MPTP- and 3-NPA-Induced Neurodegeneration as Models, Moussa B. H. Youdim, Gopal Krishna, and Chuang C. Chiueh. Index.

This chapter focuses on two models of cerebral ischemia: embolism and ligation of the middle cerebral artery (MCA). Advantages and disadvantages of MCA embolism and ligation are discussed in relation to establishing an appropriate model of stroke. In addition, the clinical relevance of each technique to the development of experimental treatment strategies is outlined, highlighting recent novel therapeutic modalities, including neural transplantation and intracerebral infusion of neurotrophic factors. Prior to discussing these two treatment strategies, an overview of the current status of pharmacologic intervention for stroke is provided, and we present critical problems (i.e., therapeutic window) inherent in drug therapy that limit its efficacy in the clinic. It is believed that the experimental evidence presented here will encourage further utilization of embolism and ligation models of cerebral ischemia in rodents as suitable animal models of stroke, and more importantly, should caution researchers and clinicians alike about critical scientific issues (i.e., early stage development of treatment strategies for stroke) that warrant validation in the laboratory setting prior to proceeding with clinical interventions.

Neural transplantation as a treatment modality for patients suffering from neurodegenerative disorders (e.g., Parkinson’s disease [PD]) has produced encouraging results. In recently conducted clinical trials, neural transplantation of human fetal ventral mesencephalic dopamine-secreting cells (the major type of cells that degenerates in PD) into the brains of PD patients has been demonstrated to ameliorate the clinical symptoms of the disease (1,2). Concrete evidence detailing clinical improvement following fetal dopaminergic cell transplantation has been reported previously in PD patients (3,4). For the first time, direct histopathological evidence became available from a transplanted PD patient, who died more than 16 mo posttransplantation of complications unrelated to the transplant procedure. Viable neural grafts were shown to integrate with the host tissue, and thus fetal tissue transplantation has been implicated as directly promoting symptomatic relief to the patient (1,2). In an attempt to circumvent logistical and ethical problems with using human fetal grafts, porcine fetal cells have been directly transplanted in PD patients and positive preliminary results have been reported (5). Other non-neural graft sources that are being examined at the preclinical level include Sertoli cells (6), carotid body cells (7), and kidney cells (8), all of which have been suggested as dopamine-or neurotrophic factor-enriched cells.

The complex etiologies and mechanisms of cell death associated with, as well as protective/reparative techniques for several neurodegenerative disorders, have been examined using animal models. We have witnessed during the last 5 yr advances in gene knockout animal models and how these animal systems can pave the way for better understanding of human diseases. For example, the discovery of the gene underlying Huntington’s disease (HD) opens the possibility that genetic therapy may be the next logical step toward finding a cure for this disease. HD is a progressive debilitating disorder associated with severe degeneration of basal ganglia neurons, especially the intrinsic neurons of the striatum, and characterized by involuntary abnormal choreiform movements and progressive dementia. In this chapter, cross-species models of HD are discussed, which recently received critical attention from researchers interested in the involvement of impaired energy metabolism in the evolution of the disease. The preceding chapters dealt largely with the utility of rodent model of 3-nitropropionic acid (3-NPA), which has been demonstrated as closely resembling the neurobiological and clinical symptoms of the disease.

Neurodegeneration can be caused by dysfunction of the mitochondrial electron transport chain (ETC), and thus a mitochondrial etiology has been suggested for many neurodegenerative disorders, such as Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD) (1,2). Accordingly, mitochondrial toxins have been used in animal models to mimic neurodegeneration. For example, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), via its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+), selectively inhibits NADH-coenzyme Q reductase (complex I) of the ETC, and induces Parkinsonism in humans, primates, and mice (3–5). Aberrant free radical formation (4), as well as impaired mitochondrial calcium metabolism (6), have been observed to succeed ETC deficits. A cascade of events leading to cell death ensues following local infusion of MPP+, and this involves increments in superoxide radicals which in combination with nitric oxide (·NO) can yield peroxynitrite anion (ONOO−) and in turn can spontaneously decompose to produce the potentially destructive reactive hydroxyl free radicals (·OH) (7,8).

Stroke remains a major brain disorder that often renders patients severely impaired and permanently disabled. There is no available treatment for reversing these deficits. Hippocampal, striatal and cortical grafting studies demonstrate that fetal cells/tissues, immortalized cells, and engineered cell lines can survive grafting into the ischemic adult brain, correct neurotransmitter release, establish both afferent and efferent connections with the host brain, and restore functional and cognitive deficits in specific models of stroke. The success of neural transplantation depends on several factors: the stroke model (location, extent, and degree of infarction), the donor cell viability and survival at pre- and post-transplantation, and the surgical technique, among others. Further exploitation of knowledge of neural transplantation therapy already available from our experience in treating Parkinson's disease needs to be critically considered for stroke therapy. While the consensus is to create a functional neuronal circuitry in the damaged host brain, there is growing evidence that trophic action of the grafts and host, as well as exogenous application of trophic factors may facilitate functional recovery in stroke. Current treatment modules, specifically that of rehabilitative medicine, should also be explored with neural transplantation therapy. However, validation of neural transplantation and any other treatment for stroke should be critically assessed in laboratory experiments and limited clinical trials. No direct treatment is recognized as safe and effective for reversing the stroke-induced brain damage and functional/cognitive deficits. The first clinical trial of neural transplantation in stroke patients is a mile-stone in stroke therapy, but subsequent large-scale trials should be approached with caution.

Cyclosporine-A (CsA) and its analogues have been shown to directly alter locomotor activity. The present study examined CsA effects on spontaneous preferential behavior in adult rats, using a two-shuttle compartment box. The initial preference (>450 s staying time) of the animal for one compartment was measured at pre-conditioning session (900 s). During conditioning session (one 60-min session per day for 6 consecutive days), the animal was alternately injected with CsA (5, 10, 20 and 30 mg/kg per day, i.p.) and vehicle, and its movement restricted to the preferred compartment and the other compartment, respectively. At post-conditioning session (900 s), animals were allowed to freely explore the box. Animals that were treated with 10 mg/kg CsA significantly spent less staying time in the preferred compartment, while those that received other CsA doses displayed a trend of decreased staying time in the preferred compartment. The present data demonstrate that CsA antagonized the spontaneous preferential behavior of animals, and warrant investigations on the drug's utility in altering other preferential behaviors (e.g. drug addiction, alcohol abuse).

Recently, we demonstrated that chronic administration of immunosuppressant drug, cyclosporine-A (CsA), does not produce impairment in memory retention of a passive avoidance task in normal adult rats. Since CsA has been used as an adjunctive therapy to avoid xenograft rejection inherent in neural transplantation therapy for neurodegenerative disorders, we replicated our previous study in animals with damaged blood-brain barrier (BBB) simulating that of the neural transplantation protocol. Adult rats with damaged BBB that received either chronic CsA (5, 10, and 20 mg/kg) or vehicle injection did not differ significantly in their memory retention of the passive avoidance task that rewarded ‘less mobile activity’, in that animals avoided electric shock when they restrained their movements within the safe compartment. General spontaneous locomotor activity also was not altered by CsA, except in animals that received 20 mg/kg, which displayed significant hypoactivity at later post-injection periods of CsA. The absence of potentiation of retention of the passive avoidance task in all CsA-treated animals, including the hypoactive ones, suggests that locomotor activity did not interfere with cognitive behavior. The present results confirm our previous findings that the therapeutic dosage (10 mg/kg) of CsA used for neural transplantation does not produce visible deleterious effects on the performance of memory retention task in immunosuppressed rats with damaged BBB.

Gender differences in the vulnerability of the lateral striatal artery (1STR artery) to systemic intoxication with 3-nitropropionic acid (3-NPA, succinate dehydrogenase inhibitor) were studied. Subcutaneous injection of 3-NPA (20 mg/kg once a day for 2 days) induced striatal selective lesions in half of male rats associated with motor symptoms (rolling, paddling, recumbency, etc) while female rats were resistant. Lesions were located in the lateral striata and characterized by astroglial necrotic cell death, enhanced immunoreaction to factor VIII-related antigen, edema, extravasation of IgG and sometimes bleeding. The motor and histological disturbances were highly sex-dependent and modulated by changes in hormonal levels. Males were more susceptible than females. Castration had little effect but ovariectomy enhanced the vulnerability. Replacement therapy with testosterone increased while estradiol or tamoxifen suppressed the vulnerability in ovariectomized females. Investigation of the arterial architecture of the brain often revealed rectangular and acute angled branchings in the centrolateral striatum where the ISTR artery feeds. A parallel in vitro toxicity study demonstrated that an extreme Ca++ overload and a strong cellular swelling resulted in astrocytic cell death. Data suggest that 1STR artery and astrocytes are highly vulnerable to 3-NPA intoxication in males. The greater vulnerability of the ISTR artery may contribute to the pathogenesis of neurodegenerative diseases, striatal bleeding, etc. Protective effects of estrogen and tamoxifen may mediate gender differences often observed in these disorders and suggest their potential use as therapeutic agents for these disorders.

We investigated the 3-nitropropionic acid (3-NP)–induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague–Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls’ posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.

Stroke remains a leading cause of death in the US, killing more than two of every five Americans (1). There are 500,000 cases of stroke each year, of which 400,000 are ischemic (2). Approximately 20% of the people who suffer a stroke die in the first week. However, more than 70% of stroke patients survive long term. There were an estimated 2,920,000 stroke survivors in 1988 (1). The economic cost of cerebrovascular disease in 1993 was estimated to be $117.4 billion, including costs of physician, nursing, hospital and nursing home services, medications, and lost productivity (3). Many patients, as well as their families, are devastated, since patients are often severely incapacitated with physical and mental disabilities. This health problem will continue to increase as the population of the US ages. It is important to rehabilitate stroke patients, so that they are as independent as possible with activities of daily living (2).

Huntington’s disease (HD) is a neurodegenerative disorder transmitted by a single autosomal dominant gene. This normally occurring Huntington gene is mutated in HD. This HD mutation involves an unstable DNA segment; the IT15 gene, which is located near the telomere of the short arm of chromosome 4, contains a trinucleotide repeat (CAG) that is expanded (> 36) and unstable (1). Clinically, HD is characterized by constant, uncontrollable choreiform movements of the body coupled with progressive deterioration of the patient’s mental status (2,3), which ultimately leads to death.

Recently, we demonstrated amelioration of behavioral deficits associated with 6-hydroxydopamine-induced hemiparkinsonism by transplanting rat testis-derived Sertoli cells into adult male rat brains. In the present study, we used adult female hemiparkinsonian rats to investigate whether the beneficial effects of transplantation of Sertoli cells may be differentially affected by gender of the animal transplant recipient. At 1 month posttransplantation, animals transplanted with Sertoli cells showed functional recovery as revealed by significant reductions in apomorphine-induced rotational behavior and asymmetrical elevated body swing behavior. Control animals that received medium alone did not display any visible behavioral recovery. These results suggest that transplantation of Sertoli cells is not male hormone-dependent and further support the use of these cells as a graft source for Parkinson's disease and other neurological disorders.

The present study proposes the use of systemic 3-nitropropionic acid (3-NP) treatment in rats as a model of Huntington's disease (HD). The systemic 3-NP model involves chronic injection of low dose intraperitoneal (i.p.) injections of 3-NP to rats once every 4 days over a period of time. Evidence from our experimental studies suggests that manipulating the number of injections can result in either increased nocturnal spontaneous locomotor activity (hyperactivity) or nocturnal akinesia (hypoactivity) [1]. For example, two injections of 3-NP (using the treatment of one injection every 4 days) result in hyperactivity, while four injections or more of 3-NP lead to hypoactivity [1]. The locomotor activity is recorded by Digiscan locomotor activity monitors [11]. The observation of these two types of locomotor activity is unique since no excitotoxin model has replicated a two-stage progression of a HD-like behavioral alteration. Most studies using excitotoxins like quinolinic acid (QA) and kainic acid (KA) have only reproduced the hyperactivity stage [4,5,7]. With the systemic 3-NP model, investigations into at least two stages of the disease are made possible. This allows for better assessment of intervention strategies such as neural transplants across different stages of the disease. The systemic 3-NP rat model is believed to be an improved animal model of HD.

The mitochondrial toxin, 3-nitropropionic acid, has been introduced in recent years as a neurotoxin that can be administered systemically to model the many neurobehavioral correlates of Huntington's disease. In this update article, we discuss some of the many findings from experiments using the systemic 3-nitropropionic model, and provide some speculative explanations supporting this model over those utilizing conventional excitotoxins or direct intrastriatal application of 3-nitropropionic acid. We infer from our own studies and those of others that the slow process of neurodegeneration, probably through apoptotic mechanism, and the progressive locomotor dysfunctions (from dyskinesia to akinesia) inherent in Huntington's disease can be accomplished by chronic, low dose systemic administration of 3-nitropropionic acid in rodents as well as in non-human primates. This 3-nitropropionic acid model offers a new venue for investigating experimental treatment strategies for Huntington's disease.

Neurological rehabilitation involves the systematic presentation of environmental stimuli and challenges that enable the patient to learn strategies for minimizing their disabilities. Rehabilitation therapy of transplant recipients may be an important factor in enhancing the efficacy of the transplanted organ or tissue to promote functional recovery. Laboratory research and clinical trials on neural transplantation, as an experimental treatment for neurological disorders (e.g., Parkinson's disease, Huntington's disease, and cerebral ischemia), have focused primarily on devising effective surgical implantation strategies with little attention devoted to the interaction between environmental factors and restorative neurosurgery. Exercise training as part of neurological rehabilitation may be an important factor for neural transplantation therapy for Parkinson's disease. Rehabilitation providers are particularly well placed to provide the environment and the support to optimize the behavioral functioning of neural transplant patients in learning to use the new grafted tissue.

We previously reported that stimulus properties of morphine can be assessed in a passive avoidance conditioning paradigm using a single dosing discrimination test. In this behavioral task, mice were trained to avoid electric footshock in a two-compartment shuttle-box by pairing morphine with shock in one compartment and saline wilh shock in the other compartment. In subsequent shock-free tests, animals can discriminate very well between saline and low doses of morphine on the one hand, and higher doses of morphine (close to that given during conditioning) on the other. Since only a single dose of morphine was presented on each test day, several days were needed to test different doses of the drug. We thus explored improving this technique by presenting different doses of morphine in a single day (cumulative dosing discrimination test). However, the animals did not dicriminate successfully between saline and morphine. Past studies have reported that mice utilize a “win-stay, lose-shift” strategy in their discrimination behavior. We hypothesized that the cumulative dosing test may have been subject to a “win-stay” bias. Accordingly, the initial exposure to the non-shock, i.e. saline, compartment promoted the animals to stay in this compartment until shock was administered. With the shock withheld throughout the test session, the “win-stay” strategy guided the animals to persevere in staying in this compartment. To confirm our hypothesis, a “lose-shift” condition through the presentation of a mild electric footshock was introduced during the test sessions. Results revealed a successful discrimination of stimulus properties of morphine, suggesting that the “lose-shift” strategy facilitated the discrimination of stimulus properties of morphine. These observations, taken together, would imply that the “win-stay, lose-shift” strategy may interact with the discrimination of the drug effects. Furthermore, the present results support the view that maintaining the environmental conditions, i.e. presence of a mild electric shock, across conditioning and test sessions can assist animals in retention of a learned task.

Cerebrovascular disease exemplifies the poor regenerative capacity of the CNS. While there are methods to prevent cerebral infarction, there is no effective therapy available to ameliorate the anatomical, neurochemical and behavioral deficits which follow cerebral ischemia. Focal and transient occlusion of the middle cerebral artery (MCA) in rodents has been reported to result in neuropathology similar to that seen in clinical cerebral ischemia. Using specific techniques, this MCA occlusion can result in a well-localized infarct of the striatum. This review article will provide data accumulated from animal studies using the MCA occlusion technique in rodents to examine whether neural transplantation can ameliorate behavioral and morphological deficits associated with cerebral infarction. Recent advances in neural transplantation as a treatment modality for neurodegenerative disorders such as Parkinson's disease, have revealed that fetal tissue transplantation may produce neurobehavioral recovery. Accordingly, fetal tissue transplantation may provide a potential therapy for cerebral infarction. Preliminary findings in rodents subjected to unilateral MCA occlusion, and subsequently transplanted with fetal striatal tissue into the infarcted striatum have produced encouraging results. Transplanted fetal tissue, assessed immunohistochemically, has been demonstrated to survive and integrate with the host tissue, and, more importantly, ameliorate the ischemia-related behavioral deficits, at least in the short term. Although, this review will focus primarily on cerebral ischemia, characterized by a localized CNS lesion within the striatum, it is envisioned that this baseline data may be extrapolated and applied to cerebral infarction in other brain areas.