Gerard H. Jansen

Expression of connexin 43 and connexin 32 gap-junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex

Abstract. The expression of the gap-junction proteins connexin (CX) 43 and 32 was evaluated in surgical specimens of brain tumors and perilesional cortex from patients with chronic medically intractable epilepsy. In human normal brain CX32 was expressed in neurons and oligodendrocytes. CX32 immunoreactivity (IR) was observed in the neuronal component of glioneuronal tumors and in all oligodendrogliomas, 50% of which showed strong labeling, independent of the grade of differentiation. CX43, normally expressed in astrocytes, was also detected in most of the human astrocytomas and in the astroglial component of glioneuronal tumors. Whereas most of the low-grade gliomas (>60%) showed strong membranous staining, most high-grade astrocytomas exhibited a reduction of the typical plasma membrane CX43-IR and intracytoplasmic localization. Immunoblot analysis showed different CX43 isoforms in control cortex and in low-grade gliomas. However, only one single isoform (corresponding to the non-phosphorylated form of CX43) appeared to be present in most high-grade gliomas. Increased expression of CX43 protein was present in reactive astrocytes in the epileptic cortex surrounding low-grade tumors as compared to control cortex, indicating the existence of a regulatory pathway involving CX43 in the reorganization of the astrocytic syncytium in regions undergoing reactive gliosis. The high expression of connexin proteins in low-grade tumors and in the peritumoral reactive astrocytes suggests that they could contribute to tumor-related seizures.

Expression of brain-derived neurotrophic factor and tyrosine kinase B receptor proteins in glioneuronal tumors from patients with intractable epilepsy: colocalization with N-methyl-D-aspartic acid receptor

Abstract. Recent evidence suggests that brain-derived neurotrophic factor (BDNF) and its tyrosine kinase B (TrkB) receptor, in addition to promoting neuronal survival and differentiation, modulates synaptic transmission by increasing N-methyl-D-aspartic acid receptor (NMDAR) activity. Overexpression of BDNF may, then, interfere with normal brain function, causing increased excitability. We have examined the immunohistochemical expression of BDNF, full-length TrkB receptor and the NMDAR subunit 1 and subunit 2A/B proteins (NMDAR1 and NMDAR2A/B) in glioneuronal tumors (gangliogliomas, GG, n=40; dysembryoplastic neuroepithelial tumors, DNT, n=15), from patients with chronic intractable epilepsy. The great majority of tumors studied were positive for all markers examined, supporting the high level of neurochemical differentiation of these lesions. BDNF and TrkB immunoreactivity (ir) was mainly observed in the neuronal component of the tumors. In GG, more than 90% of tumors contained very intense BDNF-ir ganglion cells. Double labeling confirmed the presence of BDNF-ir and TrkB-ir in neurons which contained NMDAR1. NMDAR2A/B intensely labeled abnormal neurons in both GG and DNT and co-localized with NMDAR1. The presence of BDNF and its receptor in the neuronal component of GG and DNT may suggest a role for this neurotrophin in the development of these lesions, preventing the death of abnormal neuronal cells. In addition, since these neurons contain both NMDAR1 and NMDAR2A/B subunits, the BDNF-TrkB pathway may also contribute through a modulation of glutamatergic transmission to the intrinsic epileptogenicity of glioneuronal tumors.

Early-Onset But Not Late-Onset Endothelin-A–Receptor Blockade Can Modulate Hypertension, Cerebral Edema, and Proteinuria in Stroke-Prone Hypertensive Rats

-The ability of endothelin receptor blockade to prevent and to treat established cerebral and renal injury was explored in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP) with the endothelin receptor subtype-A antagonist A127722. SHRSP were subjected to 1% NaCl intake. The start of treatment with A127722 (35 and 70 mg. kg-1. d-1, respectively) was either synchronized with salt loading or initiated after the first observation of cerebral edema with T2-weighted magnetic resonance imaging. In untreated control animals median survival was 54 days (range, 32 to 80 days) after the start of salt loading. Early-onset A127722 treatment increased median survival to 233 days (range, 92 to 407 days; P<0.05 versus controls) with 35 mg/kg and to 124 days (range, 97 to 169 days; P<0.05 versus control) with 70 mg/kg. The development of cerebral edema was prevented, and systolic blood pressure and proteinuria were dose-dependently reduced. However, all rats in the 70-mg/kg treatment group developed hemorrhages in the basal ganglia shortly before death. Late-onset A127722 treatment failed to affect survival, systolic blood pressure, or proteinuria. Nevertheless, cerebral edema was reduced but not as well as in early-onset treatment. Development of hypertension, cerebral edema, and proteinuria was prevented in SHRSP when A127722 treatment was initiated at the start of salt-loading. However, A127722 treatment did not prolong survival in SHRSP with cerebral edema. This suggests that in SHRSP the endothelin A receptor participates actively in the development of increased blood pressure and initiation of organ damage but participates minimally in established malignant hypertension and progression of target-organ damage.

Enalapril Prevents Imminent and Reduces Manifest Cerebral Edema in Stroke-Prone Hypertensive Rats

BACKGROUND AND PURPOSE Stroke-prone spontaneously hypertensive rats (SHRSP), subjected to high NaCl intake, show severe hypertension, organ damage, and early death. Preventive treatment with an angiotensin-converting enzyme (ACE) inhibitor is known to reduce mortality. Previously we found that proteinuria always precedes cerebral edema in SHRSP. Hence, in this study ACE inhibition was started later, ie, directly after manifestation of either proteinuria or cerebral edema. METHODS SHRSP were subjected to 1% NaCl intake. Group 1 served as a control. In group 2 early-onset treatment with the ACE inhibitor enalapril was initiated after proteinuria was >40 mg/d. In group 3 late-onset ACE inhibition was started after the first observation of cerebral edema with T2-weighted MRI. Cerebral edema was expressed as the percentage of pixels with an intensity above a defined threshold. RESULTS In controls median survival was 54 days (range, 32 to 80 days) after start of salt loading. The terminal level of cerebral edema was 19.0+/-3.0%. Under early-onset enalapril, median survival increased to 320 days (range, 134 to 368 days; P<0.01 versus group 1). Cerebral edema was prevented in all but 1 rat. Systolic blood pressure was slightly and transiently reduced at day 14. Proteinuria was markedly reduced (52+/-7 versus 190+/-46 mg/d in group 1 at day 7; P<0.05). Under late-onset enalapril, median survival was 264 days (range, 154 to 319 days; P<0.01 versus group 1). Cerebral edema decreased to baseline levels (9.6+/-2.9 at day 0 to 3.4+/-0.5% at day 3; (P<0.05). Ultimately cerebral edema reoccurred in 6 of the 8 rats. SBP decreased slightly at day 7 only. Proteinuria decreased from 283+/-27 at day 0 to 116+/-22 mg/d at day 7 (P<0.05). Complete remission of the original locus of cerebral edema was confirmed histologically. CONCLUSIONS In SHRSP with proteinuria, treatment with an ACE inhibitor both prevented the development of cerebral edema and reduced manifest cerebral edema and proteinuria. Survival was markedly prolonged. These findings support the use of ACE inhibition for treatment in hypertensive encephalopathy.

Treatment of combined hyperlipidemia with fluvastatin and gemfibrozil, alone or in combination, does not induce muscle damage.

Although combination therapy using 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co-A) reductase inhibitors and fibrates is efficacious in combined hyperlipidemia, such treatment has been associated with myopathy. For this reason, we studied the effects of fluvastatin and gemfibrozil, alone or in combination, on muscle. A total of 21 patients with combined hyperlipidemia were recruited who were matched for age, body mass index, and baseline levels of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglycerides, creatine phosphokinase, and myoglobin. Patients were randomized to three groups for 6-week treatment with fluvastatin at 40 mg/day, gemfibrozil at 600 mg twice daily, or a combination of the two drugs. Parameters for muscle damage were rises in levels of serum creatine phosphokinase and myoglobin compared with pre-exercise levels; these were assessed 1 hr and 8 hr after a 45 min lean body mass standardized ergometer test, which was performed before and after treatment in all patients. Biopsies from the quadriceps muscle were taken 48 hr after each test. Fluvastatin lowered total cholesterol and LDL-C by 23% and 35%, respectively (p < 0.01), with no effects on triglycerides and HDL-C. Gemfibrozil lowered triglycerides by 40% (p < 0.01) but did not lower total cholesterol or LDL-C significantly. The combination therapy decreased total cholesterol, LDL-C, and triglycerides by 28%, 29%, and 39%, respectively (p < 0.05). Pre-exercise creatine phosphokinase and myoglobin levels were not affected by treatment in any group.(ABSTRACT TRUNCATED AT 250 WORDS)

Progressive idiopathic axonal neuropathy

Abstract.Patients with a progressive disabling idiopathic axonal neuropathy could have a potentially treatable immune mediated neuropathy. To evaluate whether progressive idiopathic axonal neuropathy could be a pathologically difficult to prove vasculitic neuropathy pathologically difficult to prove or if it could be a separate clinical entity (i. e. with the axon as the primary immunological target), we performed a comparative clinical and histopathological study in 10 patients with progressive idiopathic axonal neuropathy, 10 patients with vasculitic neuropathy, and 12 patients with chronic idiopathic axonal polyneuropathy (CIAP). The clinical features and disease course in patients with progressive idiopathic axonal neuropathy and patients with vasculitic neuropathy were similar. Six patients with progressive idiopathic axonal neuropathy had been treated with prednisone and/or intravenous immunoglobulin. Disability decreased in all these six patients, but also in two of the four non-treated patients. Upon reviewing the sural nerve biopsy specimens, vasculitis was found in one patient with progressive idiopathic axonal neuropathy. Vasculitis-associated signs of ischemic injury or inflammation (most notably: large variation in fascicular axonal degeneration, perivascular inflammation, inflammation of the blood vessel wall without lumen obstruction) were found in four patients with progressive idiopathic axonal neuropathy, in all patients with vasculitic neuropathy, but were absent in patients with CIAP. The findings show that there is a small chance of finding sural nerve vasculitis upon scrutinising biopsy examination in progressive idiopathic axonal neuropathy. The presence of vasculitisassociated signs in progressive idiopathic axonal neuropathy suggests that some of these patients could have vasculitic neuropathy, even if vasculitic lesions cannot be demonstrated. However, if inflammatory changes cannot be demonstrated this does not preclude an immune-mediated origin.

Proteinuria Precedes Cerebral Edema in Stroke-Prone Rats A Magnetic Resonance Imaging Study

BACKGROUND AND PURPOSE Stroke-prone spontaneously hypertensive rats (SHRSP) subjected to high sodium intake develop severe hypertension, cerebral edema, and proteinuria, culminating in organ damage and early death. MRI, which can be applied serially, provides the unique opportunity to study temporal and quantitative relations between these changes and whether diminution of sodium intake can attenuate established cerebral edema. METHODS SHRSP were subjected to 1% NaCl in drinking water. Cerebral MRI, proteinuria and systolic blood pressure (SBP) were measured serially. After detection of cerebral edema (T2-weighted MRI), 6 rats were killed for histology, to confirm the diagnosis of cerebral edema. The others were followed up for 7 more days while salt loading was continued (n = 10, group 1) or after sodium intake was normalized (n = 7, group 2). RESULTS SHRSP invariably developed cerebral edema in 30 days (range, 8 to 54 days). At this point neurological signs were absent in 16 of 23 rats. SBP rose until 1 week before detection of cerebral edema, and then stabilized at approximately 265 mm Hg. Proteinuria invariably preceded cerebral edema, with a concentration exceeding 40 mg/d predicting development of cerebral edema in 9 days (range, 3 to 15 days). There was linear correlation (R=.62, P<.0001) between proteinuria and cerebral edema (pixels with an intensity above a defined threshold). Rats in group 1 showed an increase in cerebral edema (from 5.8+/-1.1% to 12.5+/-2.8%; P<.05), and proteinuria remained high (from 305+/-44 to 338+/-29 mg/d); and 2 died spontaneously. Rats in group 2 showed no significant change in edema (from 4.9+/-0.5% to 6.9+/-1.3%) but a marked fall in proteinuria (from 294+/-24 to 119+/-10 mg/d; P<.05), both significantly different from group 1 (P<.05); all survived. SBP remained unaltered in both groups. CONCLUSIONS Our data establish MRI as a sensitive method for detection of cerebral edema, often prior to neurological signs, in SHRSP. Proteinuria predicts cerebral edema, and these two variables, both obtained noninvasively, are quantitatively related. Moreover, in SHRSP normalizing sodium intake after salt loading attenuates development of cerebral edema and reduces proteinuria.

Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients

RATIONALE Altered expression of glutamate transporter EAAT2 protein has been reported in the hippocampus of patients with temporal lobe epilepsy (TLE). Two alternative EAAT2 mRNA splice forms, one resulting from a partial retention of intron 7 (I7R), the other from a deletion of exon 9 (E9S), were previously implicated in the loss of EAAT2 protein in patients with amyotrophic lateral sclerosis. METHODS By RT-PCR we studied the occurrence of I7R and E9S in neocortical and hippocampal specimens from TLE patients and non-neurological controls. RESULTS Both splice forms were found in all neocortical specimens from TLE patients (100% I7R, 100% E9S). This was significantly more than in controls (67% I7R, 60% E9S; P < 0.05). We also detected I7R and E9S in all seven motor cortex post-mortem samples from patients with amyotrophic lateral sclerosis. Within the TLE patient group, both splice variants appeared significantly more in non-sclerotic (100%), than in sclerotic hippocampi (69%, P < 0.05). CONCLUSION These data indicate that the epileptic brain, especially that of TLE patients without hippocampal sclerosis, is highly prone to alternative EAAT2 mRNA splicing. Our data confirm that the presence of alternative EAAT2 splice forms is not disease specific.

A novel germline mutation of PTEN associated with brain tumours of multiple lineages

We have identified a novel germline mutation in the PTEN tumour suppressor gene. The mutation was identified in a patient with a glioma, and turned out to be a heterozygous germline mutation of PTEN (Arg234Gln), without loss of heterozygosity in tumour DNA. The biological consequences of this germline mutation were investigated by means of transfection studies of the mutant PTEN molecule compared to wild-type PTEN. In contrast to the wild-type molecule, the mutant PTEN protein is not capable of inducing apoptosis, induces increased cell proliferation and leads to high constitutive PKB/Akt activation, which cannot be increased anymore by stimulation with insulin. The reported patient, in addition to glioma, had suffered from benign meningioma in the past but did not show any clinical signs of Cowden disease or other hereditary diseases typically associated with PTEN germline mutations. The functional consequences of the mutation in transfection studies are consistent with high proliferative activity. Together, these findings suggest that the Arg234Gln missense mutation in PTEN has oncogenic properties and predisposes to brain tumours of multiple lineages.

A grading system for hippocampal sclerosis based on the degree of hippocampal mossy fiber sprouting

Abstract. In patients suffering from temporal lobe epilepsy (TLE) a highly variable degree of hippocampal sclerosis (HS) can be observed. For standard neuropathological evaluation after hippocampal resection, neuronal cell loss in the hippocampal subareas is assessed (Wyler score 0–4) [Wyler et al. (1992) J Epilepsy 5: 220–225]. Other marked morphological changes in the sclerotic hippocampus are gliosis and loss of mossy fibers in the hilus and mossy fiber sprouting in the supragranular layer. In this study we quantified changes in mossy fiber density using Timms stain in resected hippocampal tissue from patients with various degrees of sclerosis. We found that tissue specimens from patients without sclerosis (W0) show almost no mossy fiber sprouting. Patients with moderate sclerosis show sprouting without fiber loss in the hilus, whereas specimens from patients with severe sclerosis show sprouting as well as fiber loss in the hilus. Thus, analysis of mossy fiber abundance in hilus and supragranular layer by the rapid and simple Timms stain is a sensitive measure for hippocampal sclerosis. It provides a reliable rapid tool for neuropathological evaluation, even if the tissue only contains dentate gyrus due to the sectioning procedure.