In vitro studies on space-conforming self-assembling silk hydrogels as a mesenchymal stem cell-support matrix suitable for minimally invasive brain application.

Advanced cell therapies require robust delivery materials and silk is a promising contender with a long clinical track record. Our aim was to optimise self-assembling silk hydrogels as a mesenchymal stem cell (MSC)-support matrix that would allow future minimally invasive brain application. We used sonication energy to programme the transition of silk (1-5% w/v) secondary structure from a random coil to a stable ß-sheet configuration. This allowed fine tuning of self-assembling silk hydrogels to achieve space conformity in the absence of any silk hydrogel swelling and to support uniform cell distribution as well as cell viability. Embedded cells underwent significant proliferation over 14 days in vitro, with the best proliferation achieved with 2% w/v hydrogels. Embedded MSCs showed significantly better viability in vitro after injection through a 30G needle when the gels were in the pre-gelled versus post-gelled state. Silk hydrogels (4% w/v) with physical characteristics matching brain tissue were visualised in preliminary in vivo experiments to exhibit good space conformity in an ischemic cavity (intraluminal thread middle cerebral artery occlusion model) in adult male Sprague-Dawley rats (n = 3). This study informs on optimal MSC-hydrogel matrix conditions for minimally invasive application as a platform for future experiments targeting brain repair.

Differential vasoactive effects of oestrogen, oestrogen receptor agonists and selective oestrogen receptor modulators in rat middle cerebral artery.

Cerebrovascular disorders are less common in pre-menopausal than post-menopausal women and in females than males. This protection may be due, in part at least, to direct effects of oestrogens on blood vessels. Oestrogen's vasodilatory mechanisms have been reported to be via the endothelium, vascular smooth muscle and extracellular matrix, depending on the vascular bed studied. Herein we investigated the vasoactive effects of oestrogen, oestrogen receptor (ER) and GPR30 agonists and selective ER modulators (SERMs) in the rat middle cerebral artery(MCA), an artery affected in focal ischaemia. MCAs isolated from male Sprague Dawley rats were mounted on a wire myograph. Concentration response curves were constructed to 17ß-oestradiol, ERα agonist-PPT, ERß agonist-DPN, GPR30 agonist-G1 and novel SERMs (LY362321 and LY2120310) in pre-constricted vessels, in the presence and absence of endothelium, blocking agents for nitric oxide synthase (L-NAME), classic ER antagonist (ICI182,780) or plasma membrane specific ERα (ERα-36) antibody. 17ß-oestradiol induced rapid vasorelaxation of the MCA which was not affected by endothelium removal, L-NAME or ICI182,780. Vasorelaxation was mimicked by PPT, DPN and G1 but not by the SERMs. Using ERα-36 antibody, effects of oestrogen were partially blocked. PPT had a greater vasorelaxation, while DPN and G1 had a lesser effect than 17ß-oestradiol. These findings indicate that activation of plasma membrane bound ERα, ß and GPR30 elicits rapid, endothelial-nitric oxide-independent relaxation of the rat MCA.

Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain.

BACKGROUND AND PURPOSE: T-cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real-time in situ brain imaging of T-cells, previously shown in models of immunological diseases, to models of experimental stroke. EXPERIMENTAL APPROACH: Male C57BL6 mice (6-8 weeks) (n= 3) received a total of 2-5 x 10(6) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled lymphocytes from donor C57BL6 mice via i.v. injection by adoptive transfer. Twenty-four hours later, recipient mice underwent permanent left distal middle cerebral artery occlusion (MCAO) by electrocoagulation or by sham surgery under isoflurane anaesthesia. Female hCD2-green fluorescent protein (GFP) transgenic mice that exhibit GFP-labelled T-cells underwent MCAO. At 24 or 48 h post-MCAO, a sagittal brain slice (1500 microm thick) containing cortical branches of the occluded middle cerebral artery (MCA) was dissected and used for multiphoton laser scanning microscopy (MPLSM). KEY RESULTS: Our results provide direct observations for the first time of dynamic T-cell behaviour in living brain tissue in real time and herein proved the feasibility of MPLSM for ex vivo live imaging of immune response after experimental stroke. CONCLUSIONS AND IMPLICATIONS: It is hoped that these advances in the imaging of immune cells will provide information that can be harnessed to a therapeutic advantage.

Imaging T-cell movement in the brain during experimental cerebral malaria.

T-cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host-parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.

The selective oestrogen receptor modulator, LY362321, is not neuroprotective in a rat model of transient focal ischaemia.

Selective oestrogen receptor modulators (SERMs) may offer improved alternatives to oestrogen as neuroprotectants in experimental stroke. The present study investigated the role of a novel SERM, LY362321, in a rat model of transient middle cerebral artery occlusion (MCAO). Female Sprague-Dawley rats were ovariectomised and began receiving daily s.c. injections of either 1 mg/kg (n = 13), 10 mg/kg (n = 14) of LY362321, or vehicle (n = 13). The left MCA was temporarily occluded (90 min), with cortical blood flow monitoring, at 12 days post ovariectomy. Sensorimotor function was assessed using a neurological score prior to the MCAO and daily for 3 days following the MCAO. Tissue was processed for infarct volume assessment using 2,3,5-triphenyltetra-zolium chloride staining. The results indicated that there were no significant differences amongst groups in cortical blood flow during the MCAO. Furthermore, there was no significant difference in infarct size amongst vehicle, 1, and 10 mg/kg treated animals: 22.9 +/- 5.0, 16.7 +/- 4.2, and 21.1 +/- 4.1, respectively, one-way anova [F(2,32) = 0.542, P = 0.587]. The MCAO induced a significant decline in neurological score in the vehicle group (from 14 to 7 at 24 h post-MCAO) but this was not significantly affected by LY362321 at either dose. In conclusion, pretreatment with a low or high dose of the novel SERM LY362321 did not significantly influence cerebral blood flow, infarct volume, or sensorimotor function in rats exposed to transient MCAO.

Oestrogen and stroke: the potential for harm as well as benefit.

Epidemiological studies point to a beneficial influence of the female reproductive hormones on stroke risk in that women have a lower incidence of stroke prior to the menopause compared with men, but this difference weakens with age and stroke risk in women rises after the menopause. However, recent Women's Health Initiative trials in post-menopausal women report an increased stroke risk on hormone replacement therapy. An influence of gender is also apparent on stroke outcome in animal models: female rats exposed to transient MCA (middle cerebral artery) occlusion sustain less brain damage than age-matched males, with loss of protection following ovariectomy. The major hormone thought to be responsible for beneficial influences on stroke incidence and outcome is oestrogen, and a large preclinical literature now exists where exogenously administered oestrogen has been studied in male and ovariectomized female rats using a range of stroke models and outcome measures. Most of these studies administer oestrogen prior to the stroke, use a model of transient ischaemia followed by reperfusion and report a significant oestrogen-induced neuroprotection. However, in some studies where the MCA is permanently occluded, oestrogen pre-treatment in ovariectomized female rats has been shown to significantly exacerbate ischaemic damage. Therefore preclinical results demonstrate harmful as well as beneficial influences of oestrogen on the ischaemic brain, highlighting the need for further study to elucidate the mechanisms responsible for both detrimental and beneficial influences. Ultimately, this could lead to the development of new classes of oestrogenic compounds with improved risk/benefit profiles, designed to selectively activate pathways inducing only the beneficial effects of oestrogen in vivo.

Brain aromatase expression after experimental stroke: topography and time course.

Brain aromatase has been shown to be increased in expression after neurotoxic damage and to exert neuroprotection via generation of local oestrogens. The present study investigates the topography and time course of brain aromatase expression after experimental stroke (middle cerebral artery occlusion (MCAO)). Ovariectomised stroke prone spontaneously hypertensive rats underwent distal MCAO by electrocoagulation. Immunohistochemistry revealed increased brain aromatase expression at 24h and 8 days in the cortical penumbra/peri-infarct zones with no increase evident at 2h or 30 days post-MCAO. Double label studies indicate that some of the increased aromatase expression is associated with astrocytic processes. Thus, this is the first evidence that aromatase protein is increased after MCAO and the location (peri-infarct), time course (within 24h) and cellular localisation (astrocytic) indicate the potential for aromatase to promote the survival of cells in the penumbra after experimental stroke by local synthesis of oestrogens.

Impaired functional recovery after stroke in the stroke-prone spontaneously hypertensive rat.

BACKGROUND AND PURPOSE: To identify if the stroke-prone spontaneously hypertensive rat (SHRSP) exhibits impaired functional recovery after stroke compared with its normotensive reference strain, the Wistar Kyoto rat (WKY). METHODS: In study 1, a 2-mm distal middle cerebral artery occlusion (middle cerebral artery occlusion) was performed in both strains and recovery assessed using a 33-point neurological score. Because SHRSPs displayed much larger infarcts than WKYs, study 2 and study 3 involved extending the length of middle cerebral artery (MCA) occlusion in the WKY to increase the volume and distribution of infarction to comparable levels with SHRSP. Animals were assessed with the neurological score, tapered beam walk, and cylinder tests. RESULTS: In study 1, infarct volume (expressed as a percent of contralateral hemisphere) was WKY 13.1+/-3% and SHRSP 19.8+/-1%. Initial neurological deficit was greater (WKY 25+/-1, SHRSP 22+/-1, out of a possible 33) and subsequent recovery was poorer in SHRSP. In studies 2 and 3, infarct volume and distribution (study 2, WKY 21.8+/-1.3%, SHRSP 22.9+/-3%; study 3, WKY 17.2+/-2%, SHRSP 16.5+/-3%) and initial neurological deficit at 2 hours after middle cerebral artery occlusion (study 2 WKY 23+/-1, SHRSP 22+/-2; study 3 WKY 25+/-1 and SHRSP 23+/-1; mean+/-SEM) were comparable between strains. However, whereas WKY recovered toward normal scores, SHRSP scored significantly lower 2 weeks (study 2) and 4 weeks (study 3) after middle cerebral artery occlusion. Beam walk data revealed long-term impairment in SHRSP contralateral limb use, compared with WKY, at days 3, 7, and 28 (P<0.05). CONCLUSIONS: SHRSP exhibit impaired functional recovery after stroke compared with WKY.

Neuroprotection by a selective estrogen receptor beta agonist in a mouse model of global ischemia.

The present study employs selective estrogen receptor (ER) agonists to determine whether 17beta-estradiol-induced neuroprotection in global ischemia is receptor mediated and, if so, which subtype of receptor (ERalpha or ERbeta) is predominantly responsible. Halothane-anesthetized female C57Bl/6J mice were ovariectomized, and osmotic minipumps containing ERbeta agonist diarylpropiolnitrile (DPN) (8 mg.kg(-1).day(-1), n = 12) or vehicle (50% DMSO in 0.9% saline) (n = 9) or ERalpha agonist propyl pyrazole triol (PPT) (2 mg.kg(-1).day(-1), n = 13) or vehicle (50% DMSO in 0.9% saline) (n = 10) were implanted subcutaneously. One week later transient global ischemia was induced by bilateral carotid artery occlusion under halothane anesthesia, and the mice were perfusion fixed 72 h later. ERbeta agonist DPN significantly reduced ischemic damage by 70% in the caudate nucleus and 55% in the CA1 region compared with vehicle controls (P < 0.05, Mann-Whitney U-statistic). In contrast, pretreatment with the ERalpha agonist PPT had no effect on the extent of neuronal damage compared with controls. The data indicate a significant estrogen receptor-mediated neuroprotection in a global cerebral ischemia model involving ERbeta.

Characterization of the microglial response to cerebral ischemia in the stroke-prone spontaneously hypertensive rat.

Stroke-prone spontaneously hypertensive rats (SHRSP) sustain more ischemic damage after middle cerebral artery occlusion than do their reference strain, the Wistar-Kyoto rat (WKY). The cause of increased stroke sensitivity is still under investigation. In general, SHRSP display a greater response to inflammatory stimuli than do WKY. Because inflammatory cells may influence the extent of damage in experimental stroke, this study has investigated the acute inflammatory response to focal ischemia in SHRSP and WKY. Adult male SHRSP (n=5) and WKY (n=5) were anesthetized and underwent distal middle cerebral artery occlusion. After 24 hours of recovery, infarct volume, neutrophil counts, and activated microglia counts were performed. SHRSP displayed more ischemic damage than did WKY (135+/-4.7 versus 102+/-4.7 mm(3) [mean+/-SEM], P<0.005). Brain neutrophil counts were extremely low in both strains. SHRSP displayed significantly more activated microglia than did WKY in the ipsilateral hemisphere (respective SHRSP versus WKY values [mean+/-SEM] were 88+/-3.6 versus 51+/-3.4 per mm(2) for the cortical peri-infarct region [P<0.005] and 183+/-7.9 versus 156+/-3.7 per mm(2) for the infarct core [P<0.05]) and in the contralateral hemisphere (eg, respective SHRSP versus WKY values were 102+/-3.2 versus 50+/-3.1 per mm(2) for the sensorimotor cortex [P<0.0001]). No neutrophils and very few activated microglia were found within the brains of naive rats. However naive SHRSP possessed more microglia (resting and activated) than did naive WKY. This study demonstrates a more pronounced microglial response to focal ischemia in SHRSP compared with WKY and provides evidence of a potential role for inflammatory processes in response to ischemic damage.

Reciprocal consomic strains to evaluate y chromosome effects.

We have previously demonstrated that the SHRSP Y chromosome contains a locus that contributes to hypertension in SHRSP/WKY F2 hybrids and that SHRSP exhibit an increased vulnerability to focal cerebral ischemia after permanent middle cerebral artery occlusion (MCAO). This increased vulnerability is inherited as a codominant trait, and a putative role for the Y chromosome has been suggested in F1 hybrids. The objective of this study was to investigate further the role of Y chromosome in blood pressure (BP) regulation and in the vulnerability to cerebral ischemia. We have constructed consomic strains by selectively replacing the Y chromosome from WKY rats with that of SHRSP, and vice versa, by using a marker-assisted breeding strategy. Permanent MCAO was carried out by electrocoagulation, with infarct volume expressed as a percentage of the ipsilateral hemisphere. Systolic blood pressure was measured by radiotelemetry during a baseline period of 5 weeks followed by a 3-week period of salt loading. We observed that the transfer of the Y chromosome from WKY onto SHRSP background significantly reduced systolic BP in consomic strains, SP.WKYGlaY(w) (n=6) versus SHRSP (n=6) (209.2+/-10.4 mm Hg versus 241.7+/-7.7 mm Hg, F=5.88, P=0.038) during the salt-loading period. In the reciprocal consomic strain, WKY.SPGlaY(s) (n=5), systolic BP was increased compared with WKY parental strain (n=6) (147.6+/-2.4 mm Hg versus 132.6+/-5.1 mm Hg, F=6.11, P=0.035) during baseline. Infarct volumes in consomic strains were not significantly different from their respective parental strain: WKY.SPGlaY(s) (n=7) versus WKY (n=7), 22.8+/-3.7% versus 22.2+/-8.0%, 95% CI=-12.7, 4.2, P=0.3; SP.WKYGlaY(w) (n=7) versus SHRSP (n=6), 37.7+/-4.4% versus 33.6+/-7.6%, 95% CI=-20.3, 12.1, P=0.5. We conclude that the SHRSP Y chromosome harbors a locus contributing to systolic BP, whereas no contribution to vulnerability to cerebral ischemia can be detected.

Investigation of estrogen status and increased stroke sensitivity on cerebral blood flow after a focal ischemic insult.

Recently the authors have shown that female stroke-prone spontaneously hypertensive rats (SHRSPs) in proestrus (high endogenous estrogen), sustain more than 20% smaller infarcts after middle cerebral artery occlusion (MCAO) compared with SHRSPs in metestrus (low endogenous estrogen). Because estrogen has vasodilator properties, the authors investigated whether the estrous state influences cerebral blood flow (CBF) after MCAO. CBF was measured 2.5 hours after a distal MCAO by [14C]iodo-antipyrine autoradiography in conscious SHRSPs either in metestrus or in proestrus. There were no significant differences in CBF when analyzed either at predetermined anatomic regions or by cumulative distribution analysis of areas with flow <25 mL/100 g/min. As a positive internal control, the authors compared results in SHRSPs with those in their normotensive reference strain, Wistar Kyoto rat. SHRSPs displayed more severe and widespread ischemia than Wistar Kyoto rats. Thus, the absence of demonstrable CBF differences between estrous states appears to be unrelated to the CBF measurement paradigm. In conclusion, the smaller infarct size afforded in proestrus in SHRSPs is unlikely to be due to an influence on CBF.

Impaired cerebral autoregulation 24 h after induction of transient unilateral focal ischaemia in the rat.

Cerebral blood flow (CBF) and cerebral autoregulation have been investigated 24 h after transient focal ischaemia in the rat. Cerebral blood flow was measured autoradiographically before and during a moderate hypotensive challenge, to test autoregulatory responses, using two CBF tracers, (99m)Tc-d,l-hexamethylproyleneamine oxide and 14C-iodoantipyrine. Prior to induced hypotension, CBF was significantly reduced within areas of infarction; cortex (28 +/- 20 compared with 109 +/- 23 mL/100 g/min contralateral to ischaemic focus, P = 0.001) and caudate (57 +/- 31 compared with 141 +/- 32 mL/100 g/min contralaterally, P = 0.005). The hypotensive challenge (mean arterial pressure reduced to 60 mmHg by increasing halothane concentration) did not compromise grey matter autoregulation in the contralateral hemisphere; CBF data were not significantly different at normotension and during hypotension. However, in the ipsilateral hemisphere, a significant volume of cortex adjacent to the infarct, which exhibited normal flow at normotension, became oligaemic during the hypotensive challenge (e.g. frontal parietal cortex 109 +/- 15% to 65 +/- 15% of cerebellar flow, P < 0.01). This resulted in a 2.5-fold increase in the volume of cortex which fell below 50% cerebellar flow (39 +/- 34 to 97 +/- 46 mm3, P = 0.003). Moderate hypotension induced a significant reduction in CBF in both ipsilateral and contralateral subcortical white matter (P < 0.01). In peri-infarct caudate tissue, CBF was not significantly affected by hypotension. In conclusion, a significant volume of histologically normal cortex within the middle cerebral artery territory was found to have essentially normal levels of CBF but impaired autoregulatory function at 24 h post-ischaemia.

Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats.

Estrogen treatment has been shown to reduce ischemic brain damage. Because endogenous estrogen levels fluctuate markedly during the estrous cycle, we investigated the effect of stage of estrous cycle on ischemic brain damage. Halothane anesthetized 3- to 5-mo-old female Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) in proestrus (high estradiol levels) or metestrus (low estradiol levels) underwent permanent middle cerebral artery occlusion. In SHRSP, infarct volume at 24 h postocclusion was 24% smaller in proestrus compared with metestrus [208.6 +/- 9.5 mm(3) (n = 7) vs. 272.7 +/- 23.8 mm(3) (n = 7), respectively, means +/- SE; P = 0.0278, unpaired t-test]. In WKY, infarct volumes were similar in proestrus and metestrus [157.0 +/- 5.4 mm(3) (n = 5) and 131.5 +/- 16.5 mm(3) (n = 8), respectively; P = not significant (NS)]. Brain swelling (ipsilateral minus contralateral hemispheric volumes) was similar in proestrus and metestrus for SHRSP [138 +/- 9 mm(3) (n = 6) and 136 +/- 10 mm(3) (n = 7), respectively] and for WKY [103 +/- 15 mm(3) (n = 5) and 90 +/- 11 mm(3) (n = 8), respectively]. Thus the reduction in infarct size in SHRSP is caused by a true attenuation of the infarct volume and not simply by a reduction in brain edema.

Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat.

We have investigated genetic transmission of increased sensitivity to focal cerebral ischemia and the influence of gender in the stroke-prone spontaneously hypertensive rat (SHRSP). Halothane-anesthetized, 3- to 5-month-old male and female Wistar-Kyoto rats (WKY), SHRSP, and the first filial generation rats (F1 crosses 1 and 2) underwent distal (2 mm) permanent middle cerebral artery occlusion (MCAO) by electrocoagulation. Infarct volume was measured by using hematoxylin-eosin-stained sections and image analysis 24 hours after ischemia and expressed as a percentage of the volume of the ipsilateral hemisphere. Infarct volume in males and females grouped together were significantly larger in SHRSP, F1 cross 1 (SHRSP father), and F1 cross 2 (WKY father), at 36.6+/-2.3% (mean+/-SEM, P<0.001, n=15), 25.4+/-2.4% (P<0.01, n=14), and 33. 9+/-1.6% (P<0.001, n=18), respectively, compared with WKY (14+/-2%, n=17). Male F1 cross 1 (18.9+/-2.4%, n=6) developed significantly smaller infarcts than male F1 cross 2 (32.8+/-2%, n=8, P<0.005). Females, which underwent ischemia during metestrus, developed larger infarcts than respective males. A group of females in which the cycle was not controlled for developed significantly smaller infarcts than females in metestrus. Thus, the increased sensitivity to MCAO in SHRSP is retained in both F1 cross 1 and cross 2 hybrids, suggesting a dominant or codominant trait; response to cerebral ischemia appears to be affected by gender and stage in the estrous cycle. In addition, the male progenitor of the cross (ie, SHRSP versus WKY) influences stroke sensitivity in male F1 cohorts.

Kainate-evoked release of adenosine from the hippocampus of the anaesthetised rat: possible involvement of free radicals.

Using microdialysis in the hippocampus of anaesthetised rats, the concentration of extracellular adenosine was estimated to be 0.8 microM. Kainic acid (0.1-25 mM) in the perfusate evoked a concentration-dependent release of adenosine with an EC50 of 940 microM. Two 5-min pulses of 1 mM kainic acid in the perfusate increased the dialysate levels with an S2/S1 ratio of 0.52 +/- 0.03. Kainate-evoked release of adenosine was reduced significantly by 10 microM tetrodotoxin and by a kappa-receptor agonist, U50, 488H (100 microM). The S2/S1 ratio was reduced by 4.5 microM 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, but not by the NMDA receptor blockers (+)-MK-801 (dizocilpine; 100 microM) or (+/-)-2-amino-5-phosphonopentanoic acid (1 mM), indicating a non-NMDA receptor-mediated process. The S2/S1 ratio was also reduced significantly by 10 mM ascorbic acid, 10 mM glutathione (a scavenger of hydroperoxides), and 1 mM oxypurinol (a xanthine oxidase inhibitor), indicating the possible involvement of free radicals. Neither the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (100 microM) nor the A1 adenosine receptor agonist R(-)-N6-(2-phenylisopropyl)adenosine (100 microM) affected release. Adenosine release evoked by kainic acid is therefore mediated by activation of non-NMDA receptors and may involve the propagation of action potentials and the production of free radicals.