Richard E. Clatterbuck

Evidence that brain-derived neurotrophic factor is a trophic factor for motor neurons in vivo

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) act upon populations of neurons that express specific receptors. The present study demonstrates that BDNF rescues motor neurons from degeneration and may also play a role in the normal physiology of these cells. BDNF is expressed in the local environment and in muscle targets of motor neurons; muscle expression is up-regulated by denervation. The alpha motor neurons express the gene encoding p145trkB, a receptor involved in BDNF signal transduction, whereas a subset of motor neurons express p75NGFR. BDNF is transported selectively to alpha motor neurons from skeletal muscles. Finally, BDNF prevents motor neuron death in the axotomized facial nucleus of the neonatal rat. The effects of BDNF on motor neurons raise the possibility that some neurotrophins may be useful in treating patients with motor neuropathies and amyotrophic lateral sclerosis.

Hemangioblastomas of the central nervous system in von Hippel-Lindau syndrome and sporadic disease.

OBJECTIVE The presentation, screening, management, and clinical outcomes of patients who presented to our institution from 1973 to 1999 with central nervous system (CNS) hemangioblastomas in von Hippel-Lindau (VHL) syndrome and sporadic disease were analyzed. METHODS The surgical pathology database of our institution was searched to identify all patients with histologically verified CNS hemangioblastomas occurring from 1973 to 1999. The medical, radiological, surgical, pathological, and autopsy records from these patients were reviewed retrospectively and statistically analyzed. RESULTS Forty patients (21 males and 19 females) presented with CNS hemangioblastomas. Twenty-five patients (62%) harbored sporadic hemangioblastomas. Fifteen patients (38%) had VHL syndrome. These 40 patients presented with 61 hemangioblastomas (8 patients had multiple lesions). Ten patients (25%) harbored spinal cord hemangioblastomas (5 patients had multiple lesions). Patients with VHL disease tended to present with neurological symptoms and signs at a younger age than patients with sporadic disease (P = 0.09), to present with multiple lesions (53%), and to develop new lesions (rate, 1 lesion/2.1 yr). Hemangioblastomas of the spinal cord were more prevalent in patients with VHL syndrome (P = 0.024). Neuroradiological screening of patients with VHL syndrome allowed identification of more than 75% of new lesions before they became symptomatic. Sixty-six surgical procedures were performed (12 patients required multiple operations). Six patients with VHL syndrome required surgery for new lesions. Surgical complications occurred in six patients (15%). Symptom resolution or arrest of progression at 1 year was documented in 88% of patients. Recurrence of symptoms from partially resected lesions occurred in eight patients (20%). No deaths associated with surgery occurred. One patient with sporadic disease and one patient with VHL syndrome (5%) died as a result of late medical complications from CNS hemangioblastomas. CONCLUSION Surgical outcomes for patients with CNS hemangioblastomas are favorable. However, management of hemangioblastomas is a more difficult and prolonged endeavor for patients with VHL syndrome. In patients with VHL syndrome, neuroradiological screening allows identification of lesions before they become symptomatic. Because patients with VHL syndrome are at risk for development of new lesions, they require lifelong follow-up.

Prevention of Experimental Cerebral Vasospasm by Intracranial Delivery of a Nitric Oxide Donor From a Controlled-Release Polymer Toxicity and Efficacy Studies in Rabbits and Rats

Background and Purpose— A reduction in the local availability of nitric oxide (NO) may play a role in the etiology of chronic cerebral vasospasm after subarachnoid hemorrhage (SAH). We investigated the toxicity and efficacy of a locally delivered NO donor from a controlled-release polymer in preventing experimental cerebral vasospasm in rats and rabbits, respectively. Methods— Diethylenetriamine/NO (DETA/NO) was incorporated into controlled release ethylene-vinyl acetate (EVAc) polymers. Twenty-eight rats were used in a dose-escalation toxicity study to establish a maximally tolerated dose of DETA/NO-EVAc polymer. In the efficacy experiment, 20 rabbits were assigned to 4 experimental groups (n=5 per group): sham operation; SAH only; SAH+empty EVAc polymer; and SAH+DETA/NO-EVAc polymer. Treatment was initiated 30 minutes after blood deposition. Basilar artery lumen patency was assessed 72 hours after hemorrhage to evaluate the efficacy of DETA/NO in preventing cerebral vasospasm. Results— In the toxicity study, a dose of 3.4 mg/kg was identified as the LD20 (dose with 20% mortality during the study period) of this DETA/NO formulation. Brain histology revealed hemorrhage and ischemic changes at the implantation site associated with high concentrations of DETA/NO. In the efficacy study, treatment with DETA/NO-EVAc polymer resulted in a significant decrease in basilar artery vasospasm compared with no treatment (93.0±4.9% versus 71.4±11.9%;P =0.035) or compared with treatment with blank EVAc polymer (93.0±4.9% versus 73.2±6.4%;P =0.003). Conclusions— Local delivery of DETA/NO prevents vasospasm in the rabbit basilar artery. Local delivery of DETA/NO via polymers is a safe and effective strategy for preventing cerebral vasospasm after SAH in this model.

The efficient calculation of neurosurgically relevant volumes from computed tomographic scans using Cavalieri's Direct Estimator.

OBJECTIVEnBecause decision making in the daily practice of neurosurgery requires judgments about changes in the volume of spatially complex three-dimensional objects, we sought a simple, rapid, and accurate method for extracting this information from standard computed tomographic (CT) scans.nnnMETHODSnVolume measurements were made by using a transparent template to overlay a grid of regularly-spaced points over CT scans of randomly selected patients with hydrocephalus or malignant gliomas. The volume of an object appearing on a scan is the product of the sum of points that fell on the object, the area associated with each point, and the distance between scan slices. This method is known as the Cavalieri Direct Estimator. We assessed the potential clinical applicability of this technique by measuring interobserver correlation for ventricular volume measurements in hydrocephalic patients, correlating brain tumor volume measurements with those obtained using the ISG Allegro system (ISG Technologies, Inc., Mississauga, Ontario) (a three-dimensional reconstruction software package), and measuring the time required for volumetric analyses.nnnRESULTSnTumor volume estimates using this method correlated highly (r = 0.999) with those made by the ISG Allegro System. Interobserver correlation for ventricular volume estimates was also high (r = 0.968). We found that the time required for application of this technique to CT scans ranged from 4 to 10 minutes.nnnCONCLUSIONSnWe conclude that the Cavalieri Direct Estimator can be easily applied to acquire volume measurements from standard CT scans and requires only a few minutes and no additional expense, making it ideal for daily use in clinical practice.

Mutations in KRIT1 in familial cerebral cavernous malformations.

OBJECTIVESnThe recognition of six unrelated Hispanic-American families in which cerebral cavernous malformations (CCM) segregated as an autosomal dominant trait established a genetic basis for this disease. Linkage analysis subsequently identified locus heterogeneity with disease genes for CCM at chromosomal regions 7q, 7p, and 3q. Recently, mutations in KRIT1, a gene on 7q at the CCM1 locus, were identified in French and Hispanic-American families with CCM. This study confirms the identity the KRIT1 founder mutation in Hispanic-Americans and reports a novel KRIT1 mutation in a Caucasian family.nnnMETHODSnOligonucleotide primers were designed to allow amplification of genomic DNA sequences from four Hispanic-American families and five non-Hispanic families for all 12 exons of the KRIT1 gene using the polymerase chain reaction (PCR). The amplified DNA was then screened using single strand conformation polymorphism analysis (SSCP) and sequencing. The expression pattern of KRIT1 was analyzed by Northern blotting.nnnRESULTSnAnalysis of the KRIT1 gene revealed a point mutation in exon 6 that predicts the substitution of a premature termination codon for glutamine at codon 248 in all four Hispanic-American families, confirming previous findings. SSCP analysis and sequencing revealed an 11 base pair duplication in exon 7 leading to a premature termination codon in one Caucasian family. Northern analysis demonstrated widespread expression of this gene, however, the highest level of expression was in the brain.nnnCONCLUSIONnThe common KRIT1 mutation causing the majority of CCM in Hispanic-Americans has been identified and independently confirmed, allowing efficient presymptomatic molecular diagnosis. In keeping with prior results, both newly identified mutations create a premature termination codon and are predicted to initiate degradation of the mutant mRNA through the nonsense-mediated mRNA decay pathway. These data strongly suggest loss of function as the relevant patho-genetic mechanism.

The prognostic value of tumor markers in patients with glioblastoma multiforme: analysis of 32 patients and review of the literature.

Although several studies have examined brain tumor markers for prognostic value, few investigations have stratified analysis based on specific histologic grade. The objective of this study was to evaluate a single histologic grade of glioma, the grade IV glioma or glioblastoma (World Health Organization Classification), with a comprehensive panel of tumor markers in an attempt to identify those with prognostic significance. Tumor samples from a cohort of patients with glioblastoma multiforme (n=32) were examined for tumor markers, DNA analysis, and clinical variables in an attempt to determine a ‘profile’ for this tumor. We used univariate and multivariate statistical analysis to determine the prognostic value of tumor cell ploidy, percent S-phase, DNA index, p53, and Ki-67 labeling index, as well as the variables of gender, race, age, location of tumor, history of chemotherapy, and primary versus recurrent tumor. Two additional tumor markers, multidrug resistance gene 1 and glutathione-S-transferase subtype pi, were included in the sample testing, but were not analyzed statistically. Univariate analysis indicated that increasing age had a strong association with decreased survival. Female gender, increasing Ki-67, no chemotherapy before sample collection, and primary glioblastoma showed some association with decreased survival in the univariate model. The univariate results indicated that race, side of tumor, ploidy, S-phase, DNA index, and p53 had no prognostic value. Multivariate modeling demonstrated that age, gender, and Ki-67 were the strongest factors associated with survival. The relevant literature is reviewed.

Interaction between krit1 and malcavernin: implications for the pathogenesis of cerebral cavernous malformations.

OBJECTIVECerebral cavernous malformations (CCM) are a relatively common autosomal dominant disorder leading to the formation of vascular malformations in the nervous system. Mutations in krit1 and malcavernin, the proteins encoded by the genes at the CCM1 and CCM2 loci, respectively, are responsible for the majority of CCMs. Similar to integrin cytoplasmic domain-associated protein-1α, a known krit1 interactor, malcavernin is a phosphotyrosine binding protein. We report here that krit1 also interacts with malcavernin. METHODSWe used two-hybrid analysis, in vivo coimmunoprecipitation, and epitope mapping to explore the interaction between krit1 and malcavernin. Immunocytochemistry was used to study the cellular localization of these proteins. RESULTSWe demonstrate that malcavernin independently binds to two of the three NPXY (asparagine, proline, undetermined/variable amino acid, and tyrosine) motifs in krit1. By immunocytochemistry, malcavernin protein is cytoplasmic at steady state, but shuttles between the nucleus and cytoplasm, despite lacking either a nuclear localization signal or a nuclear export signal in its sequence. CONCLUSIONThese data suggest that krit1 interacts with malcavernin through its NPXY motifs and may shuttle it through the nucleus via its nuclear localization signal and nuclear export signals, thereby regulating its cellular function.

Ciliary neurotrophic factor stimulates the expression of glial fibrillary acidic protein by brain astrocytes in vivo

Ciliary neurotrophic factor is a cytokine that has effects on neuronal survival and phenotype in vitro and in vivo. Ciliary neurotrophic factor has also been shown to have effects on microglia and oligodendrocytes in vitro and in vivo. In this study, we demonstrate in vivo effects of ciliary neurotrophic factor on astrocytes in both the injured and uninjured central nervous system. Ciliary neurotrophic factor increases the expression of glial fibrillary acidic protein and induces concomitant morphological changes in central nervous system astrocytes. Messenger RNA for both ciliary neurotrophic factor and the α‐component of the ciliary neurotrophic factor receptor is demonstrated in the optic nerve, an essentially pure population of central nervous system glia. We also report here that the promoter region of the glial fibrillary acidic protein gene contains sequences thought to confer direct ciliary neurotrophic factor modulation of glial fibrillary acidic protein gene transcription. Although it is thought that astrocytes are a source of endogenous ciliary neurotrophic factor in the central nervous system and that neurons express the α‐component of the ciliary neurotrophic factor receptor, the results of the present investigation suggest that astrocytes themselves respond to ciliary neurotrophic factor and that ciliary neurotrophic factor may also be important in glial cell‐cell interactions.

Neurotrophic Strategies for Treating Alzheimer's Disease: Lessons from Basic Neurobiology and Animal Modelsa

Because neurotrophic factors can prevent natural and experimental cases of neural cell death and induce and maintain differentiation, they are especially attractive agents for the treatment of neurodegenerative diseases, such as Alzheimers disease (AD). The present report argues for the specific role of particular families of trophic factors, such as neurotrophins (e.g., nerve growth factor [NGF]) and neurokines (e.g, ciliary neurotrophic factor [CNTF]), for the promotion of the survival and phenotype of subsets of central nervous system (CNS) neurons vulnerable in AD, such as basal forebrain cholinergic neurons and cortical projection neurons. Although there is ample evidence for the therapeutic role of NGF in experimental or natural injury of cholinergic neurons, not enough progress has been made on trophic models involving cortical neurons. Further understanding of the mechanisms of cell death in AD and elucidation of the transduction cascades of trophic factors will undoubtedly refine our current concepts of a neurotrophic treatment for AD.

A surgical technique for safely placing a drug delivery catheter into the pons of primates: preliminary results of carboplatin infusion.

OBJECTIVEWe sought to develop a neurosurgical procedure to access the pons with a drug delivery device for chronic therapy and collect preliminary data on the toxicity of direct infusions of carboplatin in primates. METHODSWe made midline incisions on five cynomolgus monkeys, identified the inion, made a burr hole 2.5 cm below the inion, and inserted a catheter through the cerebellum into the roof of the pons. Pumps that infused saline for 90 days or carboplatin solutions for 30 to 35 days at 10 &mgr;l/d were placed subcutaneously in the low cervical/high thoracic region. Monkeys were assessed by computed tomography and magnetic resonance imaging, laboratory studies, daily neurological observation, postmortem examinations, and histopathology. RESULTSMonkeys infused with saline and 82 &mgr;g of carboplatin remained neurologically intact throughout the infusion periods. Serial imaging showed that the catheter tip was in the pons and revealed no evidence of hemorrhage, edema, or migration. Two monkeys infused with up to 850 &mgr;g of carboplatin showed hyperintense magnetic resonance imaging signals at Days 15 and 18 and neurological deficits at approximately Week 3. Platinum levels greater than 10 ng/mg tissue were detected over a distance of 1 cm in tissue slices. Histopathology demonstrated significant tissue necrosis around the tip of the catheter. CONCLUSIONThe pons of monkeys is safely accessed with a catheter for drug delivery by using a posterior midline approach. Clinical observations, radiographic imaging, and laboratory tests of animals infused with saline for 3 months or 0.26 mg/ml of carboplatin for 1 month were unremarkable. Neurotoxicity was seen with dose levels of 2.6 mg/ml of drug for 1 month. This procedure offers opportunities for examining the toxicity of brainstem antitumor therapy in primates.