Paul J. Camarata

Sustained release of nerve growth factor from biodegradable polymer microspheres.

Although grafted adrenal medullary tissue to the striatum has been used both experimentally and clinically in parkinsonism, there is a definite need to augment long-term survival. Infusion of nerve growth factor (NGF) or implantation of NGF-rich tissue into the area of the graft prolongs survival and induces differentiation into neural-like cells. To provide for prolonged, site-specific delivery of this growth factor to the grafted tissue in a convenient manner, we fabricated biodegradable polymer microspheres of poly(L-lactide)co-glycolide (70:30) containing NGF. Biologically active NGF was released from the microspheres, as assayed by neurite outgrowth in a dorsal root ganglion tissue culture system. Anti-NGF could block this outgrowth. An enzyme-linked immunosorbent assay detected NGF still being released in vitro for longer than 5 weeks. In vivo immunohistochemical studies showed release over a 4.5-week period. This technique should prove useful for incorporating NGF and other growth factors into polymers and delivering proteins and other macromolecules intracerebrally over a prolonged time period. These growth factor-containing polymer microspheres can be used in work aimed at prolonging graft survival, treating experimental Alzheimers disease, and augmenting peripheral nerve regeneration.

“Brain Attack”: The Rationale for Treating Stroke as a Medical Emergency

Stroke is the third leading cause of death in the United States, behind only heart disease and cancer. With an estimated three million survivors of stroke in the United States, the cost to society, both directly in health care and indirectly in lost income, is staggering. Despite recent advances in basic and clinical neurosciences, which have the potential to improve the treatment of acute stroke, the general approach to the acute stroke patient remains one of therapeutic nihilism. Most basic science studies show that to be effective, acute intervention to reperfuse ischemic tissue must take place within the first several hours, as is the case with ischemic myocardium. In addition, most neuroprotective agents must also be administered within a short time frame to be effective at salvaging at-risk tissue. Recent studies have suggested that the outcome after intracerebral and subarachnoid hemorrhage is improved with early intervention. However, most stroke patients fail to present to medical attention within this short window of opportunity. The publics knowledge about stroke is woefully inadequate. However, clinicians who deal with stroke can use the dramatic changes in the treatment of acute myocardial infarction over the last 2 decades as a guide for shaping changes in the management of acute stroke. Comprehensive educational efforts aimed at clinicians and the public at large have dramatically reduced the time from symptom onset to presentation and treatment for acute myocardial infarction, enabling treatment methods such as thrombolysis to be effective. The Decade of the Brain offers a unique opportunity to all concerned with the treatment of the patient with acute stroke to engage in a concerted effort to bring patients with a brain attack to specialized neurological attention within the same timeframe that the heart attack patient is handled. Such an effort is justified because, although at the present time there are few therapeutic interventions of proven value in the treatment of acute stroke, there is more than sufficient suggestive evidence that a number of approaches may be beneficial within the first few hours after the onset of the stroke.

Continual intracavitary administration of amphotericin B as an adjunct in the treatment of aspergillus brain abscess: case report and review of the literature.

Aspergillus brain abscess is often a fatal disease, regardless of the mode of therapy. Most often seen in the compromised host, it is notoriously refractory to systemic antifungal agents and intrathecal antimycotics. Even with radical surgical debridement, only 13 patients, including the present case, have survived longer than 3 months after being treated for aspergillus brain abscess or granuloma. Studies have shown poor penetration of amphotericin B into the brain and cerebrospinal fluid. One way to achieve therapeutic levels of the agent near the abscess is through the direct introduction of the agent into the abscess site via an indwelling catheter. In the present case, a woman with an aspergillus abscess of the left temporal lobe was treated by a combination of systemic agents, radical debridement, and local therapy, resulting in a cure with a follow-up of 6 years. This is the first reported instance of the use of long-term, local antifungal therapy delivered to the area of the abscess cavity, using a closed reservoir system, and this patient is only the second renal transplant patient reported to have survived aspergillus brain abscess. This form of treatment produced no untoward long-term side effects or neurological sequelae. Local irrigation with antifungal agents should be considered in conjunction with systemic antifungal drugs and drainage and/or debridement in cases of fungal intracerebral aspergilloma. This technique may also prove useful with other fungal brain lesions.

Continual Intracavitary Administration of Amphotericin B as an Adjunct in the Treatment of Aspergillus Brain Abscess

Aspergillus brain abscess is often a fatal disease, regardless of the mode of therapy. Most often seen in the compromised host, it is notoriously refractory to systemic antifungal agents and intrathecal antimycotics. Even with radical surgical debridement, only 13 patients, including the present case, have survived longer than 3 months after being treated for aspergillus brain abscess or granuloma. Studies have shown poor penetration of amphotericin B into the brain and cerebrospinal fluid. One way to achieve therapeutic levels of the agent near the abscess is through the direct introduction of the agent into the abscess site via an indwelling catheter. In the present case, a woman with an aspergillus abscess of the left temporal lobe was treated by a combination of systemic agents, radical debridement, and local therapy, resulting in a cure with a follow-up of 6 years. This is the first reported instance of the use of long-term, local antifungal therapy delivered to the area of the abscess cavity, using a closed reservoir system, and this patient is only the second renal transplant patient reported to have survived aspergillus brain abscess. This form of treatment produced no untoward long-term side effects or neurological sequelae. Local irrigation with antifungal agents should be considered in conjunction with systemic antifungal drugs and drainage and/or debridement in cases of fungal intracerebral aspergilloma. This technique may also prove useful with other fungal brain lesions.

Optimal Timing of Hemodilution for Brain Protection in a Canine Model of Focal Cerebral Ischemia

BACKGROUND AND PURPOSEnHemodilution is known to ameliorate the effects of focal ischemia when used shortly after cerebral arterial occlusion; however, it remains to be proved whether hemodilution will be effective when used at more clinically relevant times, ie, with some delay between the onset of ischemia and initiation of therapy.nnnMETHODSnThirty-two dogs were selected for inclusion in this study. Cerebral infarction was induced by permanent occlusion of the middle cerebral and the azygos anterior cerebral arteries. The animals were allocated to 1 of 4 groups of eight animals each: arterial occlusion without hemodilution (group 1); hemodilution immediately after occlusion (group 2); hemodilution 3 hours after occlusion (group 3); and hemodilution 6 hours after occlusion (group 4). Isovolemic hemodilution to a hematocrit of 30% was performed. The animals were killed 6 days after induction of ischemia, and the infarct size was determined.nnnRESULTSnGroups 2 and 3 showed significant reduction of infarct size (P < .0001) when compared with group 1. The neurological grade of group 3 on postoperative days 4, 5, and 6 was significantly better than those of groups 1 and 4 (P < .01). Group 4 showed a significant increase in the incidence of hemorrhagic infarction when compared with groups 1 and 2 (P < .01).nnnCONCLUSIONSnThe current study indicates that hemodilution administered as much as 3 hours after ischemia is effective in reducing infarct size and improving neurological status. When administered 6 hours after ischemia, hemodilution is not helpful and may be harmful.

Neuronal Protection from Cerebral Ischemia by Synthetic Fibronectin Peptides to Leukocyte Adhesion Molecules

Leukocytes play an important role in the development of ischemia/reperfusion injury. Recent work in our laboratory has demonstrated that a mixture of synthetic fibronectin peptides to leukocyte adhesion molecules reduces ischemic brain damage after transient focal cerebral ischemia. The purpose of this study was to evaluate the efficacy of the individual peptides on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Thirty-five animals were divided into five groups: transient ischemia without treatment (Group I), treatment with arginyl-glycyl-aspartic acid (RGD) peptide (Group II), connecting segment (CS)-1 peptide (Group III), fibronectin (FN)-C/H-V peptide (Group IV), and scrambled FN-C/H-V peptide (Group V). Groups III and IV showed a significant decrease in the degree of leukocyte infiltration in the lesion and in the infarct size (p < 0.05) when compared to Groups I, II, and V. The neurological grade of Groups III and IV was significantly better than in Groups I, II, and V at 48 h after reperfusion (p < 0.01). Thus, in addition to demonstrating the potential efficacy of synthetic peptides as therapeutic agents for ischemia-reperfusion, these results also offer new insights into the mechanisms of leukocyte arrest and recruitment in ischemia/reperfusion injury.

Laminin Peptide Ameliorates Brain Injury by Inhibiting Leukocyte Accumulation in a Rat Model of Transient Focal Cerebral Ischemia

Postischemic cerebral inflammation has been reported to contribute to ischemic brain damage. During inflammation, constituents of the extracellular matrix such as fibronectin and laminin are recognized by certain integrins or proteoglycans and play an important role in the cell adhesion process. The purpose of this study was to evaluate the efficacy of peptides derived from laminin on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Forty-four animals were included in this study: transient ischemia without treatment (Group I), treatment with TG-1 peptide (Group II), GD-1 peptide (Group III), and GD-6 peptide (Group IV). Group II showed a significant reduction of the leukocyte accumulation (p < 0.001) and infarct size (p = 0.015) when compared with Group I. The neurological grade of Group II was also significantly better than in Group I at 48 h after reperfusion (p = 0.012). Based on these data, which are the first to explore the therapeutic potential of this peptide in cerebral ischemia, laminin peptide may offer a novel therapeutic approach to allaying injury in ischemic stroke.

Antagonism of leukocyte adherence by synthetic fibronectin peptide V in a rat model of transient focal cerebral ischemia

OBJECTIVEnActivated polymorphonuclear leukocytes (PMNs) seem to be directly involved in potentiating ischemic brain injury. Recent work in our laboratory demonstrated that synthetic fibronectin peptides significantly inhibit PMN accumulation in ischemic tissue, reduce the size of infarction, and reduce neurological dysfunction after transient focal cerebral ischemia in rats. The purpose of this study was to examine any dose-related effects (Experiment 1) and the optimal timing of the administration (Experiment 2) of synthetic fibronectin peptide V (FN-C/H-V) to further substantiate the role of the peptide in ameliorating cerebral ischemic damage.nnnMETHODSnFifty-six animals were included in the study. We evaluated the efficacy of FN-C/H-V on PMN accumulation in ischemic tissue, infarct size, and neurological outcomes in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion.nnnRESULTSnIn Experiment 1, the animals receiving FN-C/H-V at a dose of 10 to 15 mg/kg of body weight per injection showed significant reduction of PMN accumulation, reduction of infarct size, and improvement of neurological outcomes at 48 hours after reperfusion compared to untreated animals (P < 0.05). In Experiment 2, the animals receiving FN-C/H-V within 3 hours after reperfusion also showed significantly better results than untreated animals (P < 0.05). Despite the treatment delay, the administration of FN-C/H-V inhibited PMN accumulation after reperfusion but did not reduce the size of infarction when administered 6 hours after reperfusion.nnnCONCLUSIONnThese data suggest that relatively late postischemic administration of FN-C/H-V is effective in brain protection after ischemia/reperfusion.

Sustained Intracerebral Delivery of Nerve Growth Factor with Biodegradable Polymer Microspheres

Publisher Summary This chapter discusses sustained intracerebral delivery of nerve growth factor (NGF) with biodegradable polymer microspheres. Prolonged administration of NGF has been demonstrated to have a therapeutic action in several models of degenerative neurological diseases, including Alzheimers disease (AD), and in augmenting the survival of adrenal medullary grafts in Parkinsons disease (PD). Penetration of the blood–brain barrier (BBB) by these protein growth factors is limited in their naturally occurring state. To increase intracerebral distribution, local drug delivery strategies have been developed, including local infusion with mechanical and osmotic pumps and local synthesis and delivery by genetically modified cells and NGF-producing tumor cells. Another approach has been the use of biocompatible, biodegradable polymers. The chapter describes the methodology for encapsulation of NGF in poly( l -lactide)coglycolide microspheres, using a modified triplephase solvent evaporation technique. It discusses in vitro and in vivo evaluation of NGF release from this polymer. The chapter explains the effects of NGF delivered from polymer microspheres on the functional outcome of adrenal medullary grafts in parkinsonian rodents.

Effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-induced hemiparkinsonism on the kinematics of a two-dimensional,multijoint arm movement in the rhesus monkey.

The effects of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) on the kinematics of two-dimensional arm movements in the primate were studied. Two rhesus monkeys were trained to move a manipulandum at various distances and directions in horizontal space from a centrally located target box. Several kinematic parameters including reaction time, and time and amplitude of peak tangential velocity were analysed. Following an extensive control evaluation period, the animals were unilaterally injected with MPTP into the internal carotid artery. The animals were restudied for up to 289 days following induction of hemiparkinsonism. Larger-amplitude movements (greater than 3.5 cm) were more severely affected than smaller amplitude movements. Both animals exhibited marked changes in the arm movements including increased time-to-peak velocity and decreased peak velocity. The degree of the kinematic changes was spatially dependent, with the decrease in velocity as well as the time-to-peak velocity being more pronounced for the larger, outward movements. Reaction time increased but showed no spatial dependency. Kinematic deficits persisted over the entire time-period studied. Also, the kinematic changes were reduced by levo-3,4 dihydroxyphenylalanine in a dose-dependent manner. Tyrosine hydroxylase immunohistochemistry documented extensive cell loss in the substantia nigra. These results show that both the timing as well as the amplitude of the velocity profiles are disrupted by MPTP consistent with the known akinesia and bradykinesia of parkinsonism. Although abnormalities were present for all directions and distances, a spatial dependency to the deficits was detected. The observation of more pronounced changes for larger, outward movements suggests a role for the basal ganglia in production of larger-amplitude movements directed away from the body.