Willem Hendrik Gispen--an interview.

This text is based on three long interviews in Utrecht University's Faculty Club, and on almost 40 years of working together in the same city, the same department, the same university.

Intraspinal administration of an antibody against CD81 enhances functional recovery and tissue sparing after experimental spinal cord injury.

We previously demonstrated that the tetraspanin protein CD81 is up-regulated by astrocytes and microglia after traumatic spinal cord injury in rats and that CD81 is involved in adhesion and proliferation of cultured astrocytes and microglia. Since these reactive glial cells contribute to secondary damage and glial scar formation, we studied the effect of local administration of an anti-CD81 antibody in experimental spinal cord injury. Adult rats were subjected to a moderate spinal cord contusion injury and treated for 2 weeks with different doses of the anti-CD81 antibody AMP1 (0.5-5 microg/h) or non-immune IgG (5.0 microg/h). A technique was developed to infuse the antibodies directly into the lesion site via an intraspinal cannula connected to a pump. Functional recovery was monitored during 8 postoperative weeks by means of the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, the BBB subscore and Grid-walk test. At the end of the study, quantitative histology was performed to assess tissue sparing. Our data showed that by itself cannulation of the lesion site resulted in minimal functional and histological impairments. Application of 0.5 microg/h AMP1 resulted in a marked functional recovery (BBB 2 points; Grid-walk 30% less errors compared to control). This recovery was accompanied by an 18% increase in tissue sparing at the lesion epicentre. No gross histological changes in glial scarring were apparent. Our data demonstrate beneficial effects of an anti-CD81 antibody on functional recovery in spinal cord injured rats and suggest that this effect is mediated through a reduction in secondary tissue loss.

Cardiopulmonary bypass and long-term neurocognitive dysfunction in the rat.

Neurologic and neurocognitive complications after cardiac surgery with cardiopulmonary bypass (CPB) have been reported repeatedly. To better understand its etiology and design protective strategies, an appropriate animal model may prove useful. Although impaired short-term neurocognitive function has been recently demonstrated after CPB in rats, the demonstration of persistent long-term neurocognitive changes would be more relevant from a clinical perspective. We hypothesized that CPB results in long-term impairment of neurocognitive performance in rats. Male rats were exposed to either 60 min of normothermic non-pulsatile CPB, using a roller-pump and a neonatal membrane oxygenator, or to cannulation only (sham animals). Long-term neurocognitive function was assessed at 4 to 7 weeks after CPB (Can test), and again after 12 weeks (Morris water maze) in both operated groups and in a non-operated control group, followed by histologic evaluation of the hippocampus. In separate groups of CPB and sham animals, we also measured TNF-alpha and IL-6 in plasma. There were no significant differences in long-term neurocognitive performance or histological outcome between the three groups. Cytokine patterns were also similar in both operated groups. We conclude that CPB did not appear to cause long-term neurocognitive dysfunction in this model of CPB in young healthy rats. The lack of long-term deficits may be due to the absence of clinically important etiologic factors such as atheromatous and gaseous embolization in this model. Similar cytokine patterns in both operated groups suggest that surgical trauma rather than exposure of blood to extra-corporeal circuit was probably responsible for the inflammatory response.

Progress testing with short answer questions.

The concept of progress testing was developed in the 1970s. Significant features of progress tests are that the content is not linked to any specific course or unit, and that it reflects the final objectives of the curriculum as a whole. The questions are taken from a broad domain and cover a range of disciplines. Furthermore, the test is taken repeatedly over a period of time, to monitor students' progress. Known progress tests all use closed format questions. In 2002-2003 the University Medical Center Utrecht initiated a progress test with short answer questions. The test consists of 40 cases, each with a clinical and a basic science short-answer key feature question. This differs from other progress tests that use close format items, but also in the philosophy of mastery level testing and in the deliberate linking of basic science concepts to clinical case vignettes. The first four executions of the test show high internal consistencies (Cronbach's alpha 0.85 to 0.87) and satisfactory item parameters. The effort of marking answers is reasonable, the effort of writing case vignettes with short-answer items is less than writing MC-items if similar test reliabilities are to be achieved.

Ovariectomy and 17beta-estradiol modulate disease progression of a mouse model of ALS.

The incidence of amyotrophic lateral sclerosis (ALS) is higher among men than women but rises in women after the menopause. Estrogens may play a protective role. Treatment with estrogens has been shown to be neuroprotective in models of several neurodegenerative diseases. We therefore determined the effect of ovariectomy on female G93A mSOD1 transgenic mice, and the effect of subsequent treatment with 17beta-estradiol (E2). Ovariectomy led to a significant acceleration of disease progression of the mice, and high-dose E2 treatment significantly delayed disease progression of ovariectomized G93A mSOD1 transgenic mice. We conclude that treatment with E2 may also delay disease progression of post-menopausal women with ALS.

The time course of ischemic damage and cerebral perfusion in a rat model of space-occupying cerebral infarction.

We aimed to establish a rat model of space-occupying hemispheric infarction to evaluate potential treatment strategies. For adequate timing of therapy in future experiments, we studied the development of tissue damage, edema formation, and perfusion over time with different MRI techniques. Permanent middle cerebral artery (MCA) occlusion was performed in 32 Fisher-344 rats. Forty-six MRI experiments including diffusion weighted (DW), T2-weighted (T2W), flow-sensitive alternating inversion recovery (FAIR) perfusion-weighted, and T1-weighted (T1W) imaging before and after gadolinium were performed at 1, 3, 8, 16, 24, and 48 h of ischemia. MCA occlusion consistently led to infarction of the complete MCA territory. Mortality was 75%. Lesion volumes as derived from apparent diffusion coefficient (ADC) and T2 maps increased to maximum values of 400+/-48 mm3 at 24 h and 420+/-54 mm3 at 48 h of ischemia, respectively. Midline shift peaked at 24 h. The area with diffusion-perfusion deficit decreased to a minimum at 24 h after onset of ischemia and perfusion of the contralateral hemisphere dropped at the same time point. Leakage of gadolinium through the blood-brain barrier in the entire infarct occurred within 3 h of ischemia. Permanent intraluminal MCA occlusion in Fisher-344 rats is an adequate model for space-occupying cerebral infarction. Rats may benefit from intervention aimed at reducing tissue shift and intracranial pressure (ICP), and at improving cerebral blood flow, if initiated before 24 h after MCA occlusion. The value of treatment modalities depending on an intact blood-brain barrier should be questioned.

Delayed decompressive surgery increases apparent diffusion coefficient and improves peri-infarct perfusion in rats with space-occupying cerebral infarction.

BACKGROUND AND PURPOSE: There is no conclusive experimental support that decompressive surgery in late stages of space-occupying cerebral infarction will improve outcome. We studied the effects of delayed decompressive surgery on the development of tissue damage, edema formation, and cerebral perfusion with different MRI techniques in a rat model of space-occupying cerebral infarction. METHODS: Permanent middle cerebral artery (MCA) occlusion was performed in 6 Fisher 344 rats. Decompressive surgery was performed 17 hours after the occlusion. Each animal was assessed before surgery and 2 and 4 hours after surgery by means, of diffusion-weighted T2-weighted, and flow-sensitive alternating inversion recovery perfusion-weighted MRI. Ischemic damage was also evaluated in hematoxylin-eosin-stained brain sections. RESULTS: Lesion volume as derived from apparent diffusion coefficient (ADC) maps decreased from 522+/-98 mm3 before to 405+/-100 mm3 (P=0.016) 4 hours after decompressive surgery, whereas lesion volume from T2 maps increased from 420+/-66 mm3 before to 510+/-92 mm3 (P=0.048) 4 hours after decompressive surgery. Midline shift decreased from 1.4+/-0.1 mm to 0.5+/-0.2 mm (P=0.001). Blood flow in the noninfarcted area of the ipsilateral hemisphere improved from 25+/-9 mL/min/100 g of tissue to 38+/-9 mL/min/100 g of tissue (P=0.035). Despite the pseudonormalization of ADC, irreversible damage was found in the entire MCA territory on histological evaluation. CONCLUSIONS: In rats with space-occupying cerebral infarction, delayed decompressive surgery leads to a decrease in lesion volume derived from ADC maps, which is probably because of an increase of extracellular water formation. There are no signs that this reflects rescue of ischemic tissue.

Zinc amplifies mSOD1-mediated toxicity in a transgenic mouse model of amyotrophic lateral sclerosis.

Transgenic mice overexpressing the human mutated form (G93A) of Cu,Zn-superoxide dismutase (mSOD1) develop motor neuron degeneration resembling amyotrophic lateral sclerosis. In vitro studies have shown that mSOD1-induced, reactive oxygen species-mediated apoptosis of motor neurons depends on the presence of copper and the relative absence of zinc. Oral intake of zinc sulphate induces the expression of metallothioneins, enzymes that decrease oxidative stress, and leads to higher serum zinc, and lower copper levels. We therefore tested the effect of chronic oral administration of zinc sulfate (0.075 and 0.375 g/kg) on disease onset and survival of mSOD1 transgenic mice. We observed that zinc sulfate, rather than prolonging survival, decreased survival. We conclude that zinc sulfate amplifies the mSOD1 transgenic mouse phenotype. This finding may shed more light on the role of zinc in mSOD1-mediated toxicity.

Sexual differences in onset of disease and response to exercise in a transgenic model of ALS.

Transgenic mice that overexpress the mutant human SOD1 gene (hSOD1) serve as an animal model for amyotrophic lateral sclerosis (ALS). Age and sex are recognized as risk factors for ALS, but physical activity remains controversial. Therefore, we investigated the effect of exercise on the phenotype of male and female hSOD1 mice. Onset of disease, progression of disease and survival were measured in low-copy and high-copy hSOD1 mice that were randomized to an exercise or sedentary group. We found that onset of disease was different for the two sexes: significantly earlier in male than in female hSOD1 mice. Exercise delayed the onset of disease in female but not in male hSOD1 mice. Also, exercise delayed the total survival time in female high-copy hSOD1 mice. Muscle morphometry and motor neuron counts were similar in all experimental groups at the end of training. Sedentary female hSOD1 mice showed more frequently irregular estrous cycles suggesting a higher estrogen exposure in exercising female mice. These results suggest a possible neuroprotective effect of female sex hormones and support the view that ALS patients should not avoid regular exercise.

Neuroprotection by the endogenous cannabinoid anandamide and arvanil against in vivo excitotoxicity in the rat: role of vanilloid receptors and lipoxygenases.

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.

The effect of tirilazad mesylate on infarct volume of patients with acute ischemic stroke.

The authors investigated whether the lack of effect of tirilazad on clinical outcome in patients with acute ischemic stroke is explained by failure of tirilazad to reduce infarct volume. Overall, tirilazad had no significant effect on infarct volume. In the subgroups of male patients and of those with a cortical infarct, tirilazad significantly reduced infarct volume. These effects were reduced to nonsignificant trends after adjustment for imbalances in baseline characteristics. In conclusion, early treatment of patients with tirilazad has no effect on infarct volume.

Differential cortico-motoneuron vulnerability after chronic mitochondrial inhibition in vitro and the role of glutamate receptors.

Chronic treatment of rat cortical slices with a relative low concentration of mitochondrial inhibitor malonate leads to cortical motoneuron (CMN) death. In the neurodegenerative disease amyotrophic lateral sclerosis (ALS) corticospinal neurons, CMNs projecting to the spinal cord, degenerate. In the present study we compared the effect of chronic mitochondrial inhibition on the survival of CMNs located in the dorsal cortical areas (including corticospinal neurons) with that on ventrally located CMNs (non-corticospinal neurons) in vitro. In the explant culture model used, the dorsally located CMNs were less vulnerable to a 2-week period of mitochondrial inhibition with malonate as compared to ventrally located CMNs. Treatment with 5 mM malonate resulted in 50% surviving CMNs in the dorsal part and only 16% in the ventral part. Neuroprotection of the CMNs could be achieved with co-administration of the non-NMDA antagonist CNQX, the NMDA antagonist MK-801, or the glutamate release inhibitor riluzole, suggesting that chronic energy shortage leads to excitotoxicity. In the dorsal cortical areas CNQX, MK-801, and riluzole had a neuroprotective effect on the CMNs, whereas in the ventral cortical areas only MK-801 was neuroprotective. The sensitivity to energy depletion and consequently excitotoxicity may be related to glutamate receptor density and subunit composition in various cortical areas, but also to the projection length and input of CMNs in vivo. The present investigation gives insight in mechanisms leading to excitotoxic cell death of CMNs and may therefore be important for the development of treatment strategies in protection and survival of cortical motoneurons in ALS.

Exogenous anandamide protects rat brain against acute neuronal injury in vivo.

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Cellular changes in motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis as revealed by monoclonal antibody Py.

Transgenic mice (G93A) carrying the human amyotrophic lateral sclerosis (ALS) linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. The affected motoneurons are characterized by the presence of cellular alterations. The antigen recognized by the monoclonal antibody Py is suggested to be associated with the neurofilamentous and microtubular elements of the cytoskeleton of specific neuron populations including the spinal motoneurons. The aim of the present study was to measure changes in the relative Py-immunoreactivity per identified Choline-Acetyl-Transferase (ChAT)-immunoreactive motoneuron during the disease progression. The relative Py-immunoreactivity of identified spinal motoneurons was measured on double stained (Py and ChAT) motoneurons using a digital imaging system coupled to an inverse microscope. A significant decrease of Py-immunoreactivity was already noted in the pre-symptomatic stages of the disease even before the onset of massive motoneuron degeneration. It is concluded that the Py-antibody detects early intracellular abnormalities related to neurodegenerative changes in spinal motoneurons of transgenic SOD1-(G93A) mice.

Neuroprotection by Delta9-tetrahydrocannabinol, the main active compound in marijuana, against ouabain-induced in vivo excitotoxicity.

Excitotoxicity is a paradigm used to explain the biochemical events in both acute neuronal damage and in slowly progressive, neurodegenerative diseases. Here, we show in a longitudinal magnetic resonance imaging study that Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the main active compound in marijuana, reduces neuronal injury in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain to elicit excitotoxicity. In the acute phase Delta(9)-THC reduced the volume of cytotoxic edema by 22%. After 7 d, 36% less neuronal damage was observed in treated rats compared with control animals. Coadministration of the CB(1) cannabinoid receptor antagonist SR141716 prevented the neuroprotective actions of Delta(9)-THC, indicating that Delta(9)-THC afforded protection to neurons via the CB(1) receptor. In Delta(9)-THC-treated rats the volume of astrogliotic tissue was 36% smaller. The CB(1) receptor antagonist did not block this effect. These results provide evidence that the cannabinoid system can serve to protect the brain against neurodegeneration.

Malonate-induced cortico-motoneuron death is attenuated by NT-4, but not by BDNF or NT-3.

Neurotrophins are promising candidates to slow the progression of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease in which spinal and cortical motoneurons selectively degenerate. In a long-term in vitro model, malonate-induced toxicity and cell death of motoneurons have been demonstrated. Here we studied the neuroprotective effect of BDNF, NT-3, and NT-4 on the cell death of cortical motoneurons in an organotypic culture model after chronic mitochondrial inhibition with malonate. Our data show that NT-4 completely prevents malonate-induced toxicity, whereas BDNF or NT-3 had no neuroprotective effect. In clinical trials for ALS, predominantly focussed on the survival of spinal motoneurons, BDNF has already been tested with disappointing results; our results suggest that NT-4 may be a better neurotrophin to prevent motoneuron loss.

CD81 and microglial activation in vitro: proliferation, phagocytosis and nitric oxide production.

CD81 (TAPA), a member of the tetraspanin family of proteins, is upregulated by astrocytes and microglia after traumatic injury to the rat central nervous system (CNS). To further understand the role of CD81 in the microglial response to injury, we analysed the functional effects of a CD81 antibody, AMP1, on cultured rat microglia. We found that AMP1 suppressed microglial proliferation in a dose-dependent manner. Furthermore, AMP1 stimulated myelin phagocytosis, probably by opsonizing the myelin. The phagocytosis of latex beads, as well as the production of nitric oxide, were not significantly influenced by AMP1. These data indicate that CD81 is involved in an important subset of microglial effector functions after CNS injury.

Reproducibility of measurements of cerebral infarct volume on CT scans.

BACKGROUND AND PURPOSE: Infarct volume is increasingly used as an outcome measure in clinical trials of therapies for acute ischemic stroke. We tested which of 5 different methods to measure infarct size or volume on CT scans has the highest reproducibility. METHODS: Infarct volume and total intracranial volume were measured with Leica Q500 MCP image analysis software, or with a caliper, on 38 CT scans of patients who participated in the Tirilazad Efficacy Stroke Study II (TESS II). The scans were performed 8 days (+/-2 days) after the onset of symptoms. The 5 methods tested were based on (1) semiautomated pixel thresholding, (2) manual tracing of the perimeter, (3) a stereological counting grid, (4) measurement of the 3 largest diameters, and (5) the single largest diameter. The measurements were performed independently by 2 observers; the first observer performed all measurements twice. RESULTS: The single largest diameter did not correlate well with infarct volume. Of the other methods, manual tracing of the perimeter of the infarct had the lowest intraobserver and interobserver variability: coefficients of variation were 8.6% and 14.1%, respectively. For total intracranial volume, manual tracing also provided the highest reproducibility: intraobserver and interobserver coefficients of variation were 3.3% and 4.9%, respectively. CONCLUSIONS: Manual tracing of the perimeter is the most reproducible method for measuring the volumes of the infarct and the total intracranial space in multicenter trials of therapies for acute ischemic stroke.

Chronic mitochondrial inhibition induces glutamate-mediated corticomotoneuron death in an organotypic culture model.

There is growing evidence that mitochondrial dysfunction is an important factor in a cascade of neurotoxic events as observed during pathogenesis of various neurodegenerative diseases. In the neurodegenerative disease amyotrophic lateral sclerosis (ALS) both spinal and cortical motoneurons degenerate, but in experimental studies most attention so far has been focussed on the spinal motoneurons. In order to study the role of mitochondrial dysfunction in the pathways leading to cortical (upper) motoneuron (CMN) death, a long-term culture system of rat cortical explants was used. CMNs were visualized by immunocytochemical labeling with antibodies directed against nonphosphorylated neurofilament, SMI-32, and for their identification we also used their location in layer V of the explant, their size, and their morphological appearance. In this model the effect of mitochondrial inhibition was studied through chronic malonate treatment. For 2 weeks, low doses of complex II inhibitor malonate were added to the cultures twice a week. The malonate-induced chronic mitochondrial inhibition resulted in a dose-dependent increase of CMN death in the slices. Neuroprotection was achieved with the NMDA antagonist MK-801 and the non-NMDA antagonist CNQX indicating the involvement of glutamate in the malonate-induced CMN death. Furthermore, our data indicate that chronic mitochondrial inhibition results in CMN death, which is mediated by glutamate excitotoxicity via both non-NMDA and NMDA receptors. In this respect the present in vitro approach may act as a model for understanding mechanisms underlying CMN death in ALS.