Daniel Zemke

Mutations in the Juxtamembrane Domain of c-KIT Are Associated with Higher Grade Mast Cell Tumors in Dogs

Mast cell tumors are among the most commonly seen tumors of the skin in dogs and are more highly aggressive than mast cell tumors of other species. Some breeds display a markedly higher incidence of mast cell tumor development than others and appear to have some genetic predisposition. Recently, mutations have been found in canine mast cell tumor tissues and cell lines within the juxtamembrane domain of the protooncogene c-KIT. In previous studies utilizing a small number of cases, no association between the presence of a mutation and the breed of dog or grade of the tumor could be identified. An expanded study with a larger sample set was performed to explore this possibility. The juxtamembrane domain of c-KIT was amplified using the polymerase chain reaction from genomic DNA preparations of 88 paraffin-embedded mast cell tumors from selected breeds. Mutations, consisting of duplications and deletions, were found in 12 of the tumors. A significant association was found between the presence of a mutation and a higher grade of tumor but not between breed and grade or between breed and the presence of a mutation.

The potential of minocycline for neuroprotection in human neurologic disease

Minocycline is a member of the tetracycline class of molecules with broad-spectrum antibiotic activity. The unique properties of minocycline result in increased tissue distribution when compared with the other tetracyclines. Of particular interest is the ability of minocycline to diffuse into the central nervous system at clinically effective levels. Aside from its antimicrobial properties, minocycline has been found to have beneficial effects on inflammation, microglial activation, matrix metalloproteinases, nitric oxide production, and apoptotic cell death. Concordantly, minocycline has been found to have neuroprotective effects in animal models of a number of diseases including stroke, multiple sclerosis, and Parkinson disease. The proven safety of minocycline over decades of use as an antibiotic suggests that it may have potential for development into an effective treatment of multiple neurologic conditions in humans.

Asiatic acid, a pentacyclic triterpene from Centella asiatica, is neuroprotective in a mouse model of focal cerebral ischemia

Asiatic acid, a triterpenoid derivative from Centella asiatica, has shown biological effects such as antioxidant, antiinflammatory, and protection against glutamate‐ or β‐amyloid‐induced neurotoxicity. We investigated the neuroprotective effect of asiatic acid in a mouse model of permanent cerebral ischemia. Various doses of asiatic acid (30, 75, or 165 mg/kg) were administered orally at 1 hr pre‐ and 3, 10, and 20 hr postischemia, and infarct volume and behavioral deficits were evaluated at day 1 or 7 postischemia. IgG (blood–brain barrier integrity) and cytochrome c (apoptosis) immunostaining was carried out at 24 hr postischemia. The effect of asiatic acid on stress‐induced cytochrome c release was examined in isolated mitochondrial fractions. Furthermore, its effects on cell viability and mitochondrial membrane potential were studied in HT‐22 cells exposed to oxygen‐glucose deprivation. Asiatic acid significantly reduced the infarct volume by 60% at day 1 and by 26% at day 7 postischemia and improved neurological outcome at 24 hr postischemia. Our studies also showed that the neuroprotective properties of asiatic acid might be mediated in part through decreased blood–brain barrier permeability and reduction in mitochondrial injury. The present study suggests that asiatic acid may be useful in the treatment of cerebral ischemia.

Carnosine Is Neuroprotective Against Permanent Focal Cerebral Ischemia in Mice

Background and Purpose— Carnosine is a naturally occurring dipeptide with multiple neuroprotective properties. In addition, it is well tolerated in high doses with minimal side effects. The purposes of this study were to determine whether carnosine is neuroprotective in permanent focal cerebral ischemia and to determine potential mechanisms of neuroprotection. Methods— We investigated the efficacy of carnosine in a mouse model of permanent focal cerebral ischemia. The effects of carnosine were investigated with respect to neuronal damage and infarct formation, endogenous antioxidant status, and matrix metalloproteinase activity. Results— Carnosine significantly decreased infarct size and neuronal damage when administered at time points both before and after the induction of ischemia. Carnosine also decreased reactive oxygen species levels in the ischemic brain, preserved normal glutathione levels, and decreased matrix metalloproteinase protein levels and activity. Conclusions— Carnosine is neuroprotective in focal cerebral ischemia and appears to influence deleterious pathological processes that are activated after the onset of ischemia.

The therapeutic potential of statins in neurological disorders.

Statins are currently among the most commonly prescribed agents for the prevention of cardiovascular disease. Statins reduce serum cholesterol levels by reversibly inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, in the nanomolar range. Mounting evidence suggests that in addition to their vascular effects such as stabilization of atherosclerotic plaques and decreased carotid intimal-medial thickness, statins have additional properties such as endothelial protection via actions on the nitric oxide synthase system as well as antioxidant, anti-inflammatory and anti-platelet effects. These effects of statins might have potential therapeutic implications in various neurological disorders such as stroke, Alzheimers disease, Parkinsons disease, multiple sclerosis and primary brain tumors. In this review, the major protective mechanisms of statins and their applicability to the treatment of neurological disease are summarized. Although further experiments are required, currently available data would seem to indicate that clinical trials to determine the safety and efficacy of statins in a number of disorders are warranted.

Differential Neuroprotective Effects of Carnosine, Anserine, and N-Acetyl Carnosine against Permanent Focal Ischemia

Carnosine (β‐alanyl‐L‐histidine) has been shown to exhibit neuroprotection in rodent models of cerebral ischemia. In the present study, we further characterized the effects of carnosine treatment in a mouse model of permanent focal cerebral ischemia and compared them with its related peptides anserine and N‐acetylated carnosine. We also evaluated the efficacy of bestatin, a carnosinase inhibitor, in ameliorating ischemic brain damage. Permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery (pMCAO). Mice were subsequently randomly assigned to receive an intraperitoneal injection of vehicle (0.9% saline), carnosine, N‐acetyl carnosine, anserine, bestatin alone, or bestatin with carnosine. Infarct size was examined using 2,3,5‐triphenyltetrazolium chloride staining 1, 3, and 7 days following pMCAO, and neurological function was evaluated using an 18‐point‐based scale. Brain levels of carnosine were measured in treated mice using high‐performance liquid chromatography 1 day following pMCAO. We demonstrated that treatment with carnosine, but not its analogues, was able to significantly reduce infarct volume and improve neurological function compared with those in vehicle‐treated mice. These beneficial effects were maintained for 7 days post‐pMCAO. In contrast, compared with the vehicle‐treated group, bestatin‐treated mice displayed an increase in the severity of ischemic lesion, which was prevented by the addition of carnosine. These new data further characterize the neuroprotective effects of carnosine and suggest that carnosine may be an attractive candidate for testing as a stroke therapy.

Characterization of an Undifferentiated Malignancy as a Mast Cell Tumor Using Mutation Analysis in the Proto-Oncogene c-KIT

A 6.5-year-old female Boxer was euthanized and presented for necropsy following rapid clinical decline concomitant with the development of numerous tumor masses. The largest of these masses was in the same location as a mast cell tumor that had been previously removed from this dog. Gross examination revealed the presence of nodules 5–200 mm in diameter throughout the body, including the lymph nodes. Histologic analysis showed an influx of round cells with no granules, leading to the provisional diagnosis of systemic lymphosarcoma. Immunohistochemical staining for B- and T-lymphocyte antigens was negative. Molecular tests were used to identify a tandem duplication in the c-KIT proto-oncogene from both the earlier mast cell tumor and the current nodules, implicating a common origin. Addition of molecular testing to conventional necropsy evaluations allowed a definitive diagnosis of mast cell tumors.

Delayed ischemia after subarachnoid hemorrhage: result of vasospasm alone or a broader vasculopathy?

The term vasospasm is commonly used to describe constriction of cerebral blood vessels after subarachnoid hemorrhage which results in the restriction of blood flow and ischemia in affected portions of the brain. The pathophysiological changes that underlie vascular constriction after subarachnoid hemorrhage include changes within the vessel walls themselves, alteration of the levels of several vasoactive substances, and broader pathological conditions such as immune responses, inflammation, and oxidative damage. In this review, we summarize the current state of knowledge concerning the processes that occur in cerebral blood vessels after subarachnoid hemorrhage and how they may be involved in the development of vasospasm. We also propose that, rather than merely vasospasm, the multitude of vascular effects occurring after subarachnoid hemorrhage can be best described as a post-subarachnoid hemorrhage vasculopathy.

The Effect of Anticonvulsant Drugs on the Fibrinolytic Activity of Tissue Plasminogen Activator

Background and Purpose: Several anticonvulsant drugs have been found to be neuroprotective in preclinical models of stroke, and such drugs may possibly be given in combination with other stroke treatments such as recombinant tissue plasminogen activator (rt-PA). The goal of this study was to test for potential interactions between rt-PA and selected anticonvulsants. Methods: A spectrophotomeric assay was used to monitor the lysis of fibrin clots in the presence of rt-PA and the drugs levetiracetam, valproic acid, phenytoin and phenobarbital. Results: The drugs tested were found to have no effect on either the rate or total amount of lysis induced by rt-PA. Conclusions: Although further studies are required in order to explore the effects of these drugs in stroke patients, the results suggest that co-administration of rt-PA and anticonvulsant drugs may be safe and viable.

The Potential of Adaptive Design in Animal Studies.

Clinical trials are the backbone of medical research, and are often the last step in the development of new therapies for use in patients. Prior to human testing, however, preclinical studies using animal subjects are usually performed in order to provide initial data on the safety and effectiveness of prospective treatments. These studies can be costly and time consuming, and may also raise concerns about the ethical treatment of animals when potentially harmful procedures are involved. Adaptive design is a process by which the methods used in a study may be altered while it is being conducted in response to preliminary data or other new information. Adaptive design has been shown to be useful in reducing the time and costs associated with clinical trials, and may provide similar benefits in preclinical animal studies. The purpose of this review is to summarize various aspects of adaptive design and evaluate its potential for use in preclinical research.