Ursula I. Tuor

Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy

Insulin deficiency in type I diabetes may lead to cognitive impairment, cerebral atrophy and white matter abnormalities. We studied the impact of a novel delivery system using intranasal insulin (I-I) in a mouse model of type I diabetes (streptozotocin-induced) for direct targeting of pathological and cognitive deficits while avoiding potential adverse systemic effects. Daily I-I, subcutaneous insulin (S-I) or placebo in separate cohorts of diabetic and non-diabetic CD1 mice were delivered over 8 months of life. Radio-labelled insulin delivery revealed that I-I delivered more rapid and substantial insulin levels within the cerebrum with less systemic insulin detection when compared with S-I. I-I delivery slowed development of cognitive decline within weekly cognitive/behavioural testing, ameliorated monthly magnetic resonance imaging abnormalities, prevented quantitative morphological abnormalities in cerebrum, improved mouse mortality and reversed diabetes-mediated declines in mRNA and protein for phosphoinositide 3-kinase (PI3K)/Akt and for protein levels of the transcription factors cyclic AMP response element binding protein (CREB) and glycogen synthase kinase 3beta (GSK-3beta) within different cerebral regions. Although the murine diabetic brain was not subject to cellular loss, a diabetes-mediated loss of protein and mRNA for the synaptic elements synaptophysin and choline acetyltransferase was prevented with I-I delivery. As a mechanism of delivery, I-I accesses the brain readily and slows the development of diabetes-induced brain changes as compared to S-I delivery. This therapy and delivery mode, available in humans, may be of clinical utility for the prevention of pathological changes in the diabetic human brain.

DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat

DNA extracted from regional brain samples of hypoxic/ischemic neonatal rats showed internucleosomal cleavage indicative of apoptosis. Cells containing cleaved DNA were identified by in situ labelling in the cortex, hippocampus, striatum and thalamus of the ipsilateral hemisphere. When the effects of increasing the length of the hypoxia were examined, increases were seen in the amount of internucleosomally cleaved DNA and in the number of labelled cells.

Dexamethasone prevents hypoxic-ischemic brain damage in the neonatal rat.

ABSTRACT: Glucocorticoid therapy is frequently used in perinatology and neonatology for its beneficial pulmonary effects. We investigated the influence of neonatal glucocorticoid administration on brain damage caused by a concurrent episode of cerebral hypoxia-ischemia. Various doses of dexamethasone in several treatment schedules were administered to 7-d-old rats that were also subjected to unilateral cerebral hypoxia-ischemia. In 79% of control rats, a large unilateral cerebral infarction occurred, whereas all rats pretreated with dexamethasone in doses of 0.01 to 0.5 mg/kg/d for 3 d had no infarction (p < 0.001). The neuroprotective effect of dexamethasone pretreatment was dose- and time-dependent. Treatment with dexamethasone after the insult or with lower doses before the insult did not prevent infarction. The neuroprotective effect was not immediate: single doses 0 to 3 h prehypoxia were not effective but a single dose 24 h before hypoxiaischemia prevented cerebral infarction. The results demonstrate that glucocorticoid administration in the neonatal period, even in low doses, protects the brain during subsequent periods of hypoxia-ischemia.

Functional magnetic resonance imaging in rats subjected to intense electrical and noxious chemical stimulation of the forepaw.

&NA; We examined whether cerebral activation to two different intense and painful stimuli could be detected using functional magnetic resonance imaging (fMRI) in &agr;‐chloralose anesthetized rats. Experiments were performed using a 9.4 T magnet and a surface coil centered over the forebrain. A set of gradient echo images were acquired and analyzed using our software based on fuzzy cluster analysis (EvIdent™). Following the injection of 50 &mgr;l of formalin (5%) into the forepaw we observed a regional increase in signal intensity in the MR images in all animals. Anterior cingulate cortex, frontal cortex and sensory‐motor cortex were some of the regions that activated frequently and often bilaterally. Surprisingly, activation appeared sequentially, often occurring first in either the right or the left hemisphere with a separation of seconds to minutes between peak activations. Morphine pre‐treatment (1 mg/kg, i.v.) delayed and/or reduced the intensity of the activation resulting in a decrease in the overall response. Following episodes of intense electrical stimulation, produced by two brief stimulations (15 V, 0.3 ms, 3 Hz) of the forepaw, activation was observed consistently in the sensory‐motor cortex contralateral to the stimulation. Activation also occurred frequently in the anterior cingulate cortex, ipsilateral sensory‐motor cortex and frontal cortical regions. All these regions of activation were markedly reduced during nitrous oxide inhalation. Treatment with morphine resulted in an inhibition of the activation response to electrical stimulation in most regions except for sensory‐motor cortex. Thus, electrical and chemical noxious stimuli activated regions that are known to be involved in the central processing of pain and morphine modified the activation observed. fMRI combined with appropriate exploratory data analysis tools could provide an effective new tool with which to study novel analgesics and their effects on the CNS processing of pain in animal models.

Correspondence of AQP4 expression and hypoxic‐ischaemic brain oedema monitored by magnetic resonance imaging in the immature and juvenile rat

Whether the water channel protein AQP4 is involved in the very early cell swelling and brain oedema observed with cerebral hypoxia–ischaemia (HI) and whether this response depends on the maturity of brain were investigated by comparing regional changes in AQP4 protein expression and signal intensity on magnetic resonance (MR) images in immature and juvenile brains. Maps of T2 and the apparent diffusion coefficient (ADC) of water were acquired in 1‐ and 4‐week‐old rats at times prior to HI, within the last 5 min of HI and 1 h or 24 h afterwards. AQP4 expression assessed with Western blotting was not significantly reduced until 24 h post‐HI irrespective of age. However, AQP4 immunostaining was decreased at the end of HI and at 1 h or 24 h after HI in the hemisphere ipsilateral to the occlusion with changes being similar in both age groups and coinciding well with regional reductions in ADC. IgG immunostaining to assess blood–brain barrier integrity and T2 were unchanged at early time points in 4‐week old rats despite decreases in AQP4 immunostaining. Thus, at early time points there were decreases in AQP4 detected with immunostaining but not Western blotting methods. However, the good correlation between alterations in ADC and AQP4 immunostaining suggests that changes in the AQP4 are involved in some of the early changes in brain water distribution observed in hypoxia‐ischemia, and supports the speculation that AQP4 is involved in the transport of water across the perivascular membranes into the vascular lumen.

MR molecular imaging of early endothelial activation in focal ischemia.

Focal ischemia followed by reperfusion initiates a harmful P‐ and E‐selectin–mediated recruitment of leukocytes in brain microvasculature. In this study, we tested whether a novel magnetic resonance (MR) contrast agent (Gd‐DTPA‐sLex A), which is designed to bind to activated endothelium could be detected by MR imaging (MRI) in a focal stroke mouse model. MRIs (9.4T) of the brain were acquired 24 hours after transient middle cerebral artery occlusion. T1 maps were acquired repeatedly before and up to 1.5 hours after the intravenous injection of either Gd‐DTPA or Gd‐DTPA‐sLex A. Analysis of images included a pixel‐by‐pixel subtraction of T1 maps from the precontrast T1 maps and quantification of T1 within the ischemic area. After injection of Gd‐DTPA‐sLex A, T1 decreased compared with precontrast levels, and an interhemispheric difference between the pre–post contrast T1 developed within the stroke lesion at a mean time of 52 minutes after injection (p < 0.05). Animals injected with Gd‐DTPA did not exhibit changes in T1 signal intensity between regions of the ipsilateral and contralateral hemispheres, indicating that the reductions in T1 observed with Gd‐DTPA‐sLex A were unrelated to blood–brain barrier breakdown. Fluorescent‐labeled sLex A administered intravenously was observed to bind to the endothelium of injured but not control brain. The study suggests that the contrast agent Gd‐DTPA‐sLex A can be used to visualize early endothelial activation after transient focal ischemia in vivo with MRI. Ann Neurol 2004;56:116–120

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke

BackgroundThe link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa).ResultsMRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice, Δ T1 stroke -Δ T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLeX) group compared to Gd-DTPA, indicative of sLeX mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice.ConclusionP-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

Prevention of hypoxic-ischemic damage with dexamethasone is dependent on age and not influenced by fasting

Pretreatment with the synthetic glucocorticoid dexamethasone prevents hypoxic-ischemic brain damage in 7-day-old neonatal rats. We presently characterize the response further by examining the effect of varying the age, the glucocorticoid, and the time of injection and by examining whether fasting can influence the response. Rats (n = 193) were randomized to one of 16 different treatment groups and subjected to hypoxia-ischemia (right carotid artery occlusion +8% O2 which was 3 h in duration for 7-day, 1 h for 2-week, and 30 min for 1-month-old animals). The brains were subsequently perfusion fixed and the area of infarction was measured from hematoxylin- and eosin-stained sections. Time dependence studies demonstrated that treatment with 0.1 mg/kg intraperitoneal dexamethasone 4 h prior to hypoxia reduced infarct size compared to vehicle-treated animals whereas pretreatment at either 48 h or 4 days was ineffective. Dexamethasone pretreatment (4 h) also provided neuroprotection against 4 h of hypoxia-ischemia. Fasted animals which received dexamethasone had reduced blood glucose levels yet markedly less damage than controls. Another glucocorticoid, methylprednisolone (0.7 mg/kg), also reduced infarction. In 2-week-old animals the area of infarction was reduced by pretreatment with dexamethasone, whereas in 1-month-old animals dexamethasone was ineffective. The results suggest that a glucocorticoid-mediated response intervenes in events leading to neuronal death in young animals but not older animals once myelination and synaptogenesis are complete.

Correlation of Cerebral Hypoxic-Ischemic T2 Changes With Tissue Alterations in Water Content and Protein Extravasation

Background and Purpose— Age-dependent changes in T2-weighted MR images have been reported in cerebral hypoxia-ischemia. However, the biophysical mechanisms responsible for the image changes remain poorly defined. We investigated whether cerebral hypoxia-ischemia–induced T2 changes correlate with alterations in either water content or protein extravasation. Methods— One- and 4-week-old rats were subjected to unilateral carotid artery occlusion plus hypoxia in 8% oxygen. T2 images were acquired before, during, and 1 or 24 hours after hypoxia-ischemia. Blood-brain barrier disruption and brain edema were evaluated by immunohistological detection of IgG extravasation and measurement of water content by dry-wet weight and specific gravity methods. Results— In 1-week-old rats, T2 values, areas of hyperintensity on T2-weighted images, and water content in the ipsilateral hemisphere increased during hypoxia-ischemia, recovered at 1 hour after hypoxia-ischemia, and increased again at 24 hours after hypoxia-ischemia. Extravasation of IgG occurred during hypoxia-ischemia and remained detectable 24 hours after hypoxia-ischemia. In 4-week-old rats, an increase in T2 or extravasation of IgG did not occur until 24 hours after hypoxia-ischemia despite a comparable elevation in water content during and soon after hypoxia-ischemia. Conclusions— T2 imaging appears reliable for detecting edema associated with disruption of the blood-brain barrier but not necessarily an increase in cerebral water or plasma proteins alone. The different hypoxic-ischemic changes in T2 in immature and older brain are associated with differences in alterations in water content plus extravasation of protein, consistent with age-dependent differences in hypoxic-ischemic alterations in vascular permeability.

Protection against Recurrent Stroke with Resveratrol: Endothelial Protection

Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier in vivo demonstrated that resveratrol treatment reduced blood-brain barrier disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro ‘ischemia’ resulting in improved viability against oxygen and glucose deprivation (39.6±6.6% and 81.3±9.5% in vehicle and resveratrol treated cells, respectively). An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.