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.

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.

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.