Bushra Wali

Progesterone attenuates hemorrhagic transformation after delayed tPA treatment in an experimental model of stroke in rats: involvement of the VEGF–MMP pathway

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for acute stroke, but its use remains limited. Progesterone (PROG) has shown neuroprotection in ischemia, but before clinical testing, we must determine how it affects hemorrhagic transformation in tPA-treated ischemic rats. Male Sprague–Dawley rats underwent middle cerebral artery occlusion with reperfusion at 4.5 hours and tPA treatment at 4.5 hours, or PROG treatment intraperitoneally at 2 hours followed by subcutaneous injection at 6 hours post occlusion. Rats were killed at 24 hours and brains evaluated for cerebral hemorrhage, swelling, blood–brain barrier (BBB) permeability, and levels of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor level (VEGF), and tight junction (TJ) proteins. We also evaluated PROGs efficacy in preventing tPA-induced impairment of transendothelial electrical resistance (TEER) and TJ proteins under hypoxia/reoxygenation in the endothelial cells. Delayed tPA treatment induced significant hemorrhagic conversion and brain swelling. Treatment with PROG plus tPA ameliorated hemorrhage, hemispheric swelling, BBB permeability, MMP-9 induction, and VEGF levels compared with controls. Progesterone treatment significantly prevented tPA-induced decrease in TEER and expression of occludin and claudin-5, and attenuated VEGF levels in culture media subjected to hypoxia. The study concluded that PROG may extend the time window for tPA administration in ischemic stroke and reduce hemorrhagic conversion.

Progesterone in experimental permanent stroke: a dose-response and therapeutic time-window study

Currently, the only approved treatment for ischaemic stroke is tissue plasminogen activator, a clot-buster. This treatment can have dangerous consequences if not given within the first 4 h after stroke. Our group and others have shown progesterone to be beneficial in preclinical studies of stroke, but a progesterone dose-response and time-window study is lacking. We tested male Sprague-Dawley rats (12 months old) with permanent middle cerebral artery occlusion or sham operations on multiple measures of sensory, motor and cognitive performance. For the dose-response study, animals received intraperitoneal injections of progesterone (8, 16 or 32 mg/kg) at 1 h post-occlusion, and subcutaneous injections at 6 h and then once every 24 h for 7 days. For the time-window study, the optimal dose of progesterone was given starting at 3, 6 or 24 h post-stroke. Behavioural recovery was evaluated at repeated intervals. Rats were killed at 22 days post-stroke and brains extracted for evaluation of infarct volume. Both 8 and 16 mg/kg doses of progesterone produced attenuation of infarct volume compared with the placebo, and improved functional outcomes up to 3 weeks after stroke on locomotor activity, grip strength, sensory neglect, gait impairment, motor coordination and spatial navigation tests. In the time-window study, the progesterone group exhibited substantial neuroprotection as late as 6 h after stroke onset. Compared with placebo, progesterone showed a significant reduction in infarct size with 3- and 6-h delays. Moderate doses (8 and 16 mg/kg) of progesterone reduced infarct size and improved functional deficits in our clinically relevant model of stroke. The 8 mg/kg dose was optimal in improving motor, sensory and memory function, and this effect was observed over a large therapeutic time window. Progesterone shows promise as a potential therapeutic agent and should be examined for safety and efficacy in a clinical trial for ischaemic stroke.

Vitamin D prevents hypoxia/reoxygenation-induced blood-brain barrier disruption via vitamin D receptor-mediated NF-kB signaling pathways.

Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)2D3 on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)2D3 treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF—kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )2D3 with the vitamin D receptor (VDR) was inhibited by pyridoxal 5’-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )2D3 against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells.

Glibenclamide Administration Attenuates Infarct Volume, Hemispheric Swelling, and Functional Impairments following Permanent Focal Cerebral Ischemia in Rats

Studies from a single laboratory have shown that in rodent models of permanent stroke, administration of the sulfonylurea glibenclamide (Glib) is highly effective in reducing edema, mortality, and lesion volume. The Stroke Therapy Academic Industry Roundtable (STAIR) recommends that new acute treatments for ischemic stroke to be replicated across different laboratories. Accordingly, we examined the effect of low-dose Glib in a permanent suture occlusion model of stroke. Male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO) followed by an initial intraperitoneal injection of Glib (10 μg/kg) and the start of a constant infusion (200 ng/h) via miniosmotic pump at the onset of ischemia. Functional deficits were assessed by Neurological Severity Score (NSS) and grip-strength meter at 24 and 48 h after pMCAO. Glib-treated rats showed a significant reduction in infarct volume, lower NSS, and less hemispheric swelling compared to vehicle. Grip strength was decreased significantly in pMCAO rats compared to shams and significantly improved by treatment with Glib. Taken together, these data indicate that Glib has strong neuroprotective effects following ischemic stroke and may warrant further testing in future clinical trials for human stroke.

Prophylactic Edaravone Prevents Transient Hypoxic-Ischemic Brain Injury Implications for Perioperative Neuroprotection

Background and Purpose— Hypoperfusion-induced thrombosis is an important mechanism for postsurgery stroke and cognitive decline, but there are no perioperative neuroprotectants to date. This study investigated whether prophylactic application of Edaravone, a free radical scavenger already used in treating ischemic stroke in Japan, can prevent infarct and cognitive deficits in a murine model of transient cerebral hypoxia-ischemia. Methods— Adult male C57BL/6 mice were subjected to transient hypoxic-ischemic (tHI) insult that consists of 30-minute occlusion of the unilateral common carotid artery and exposure to 7.5% oxygen. Edaravone or saline was prophylactically applied to compare their effects on cortical oxygen saturation, blood flow, coagulation, oxidative stress, metabolites, and learning-memory using methods that include photoacoustic imaging, laser speckle contrast imaging, solid-state NMR, and Morris water maze. The effects on infarct size by Edaravone application at different time points after tHI were also compared. Results— Prophylactic administration of Edaravone (4.5 mg/kg×2, IP, 1 hour before and 1 hour after tHI) improved vascular reperfusion, oxygen saturation, and the maintenance of brain metabolites, reducing oxidative stress, thrombosis, white-matter injury, and learning impairment after tHI insult. Delayed Edaravone treatment after 3 h post-tHI became unable to reduce infarct size. Conclusions— Acute application of Edaravone may be a useful strategy to prevent postsurgery stroke and cognitive impairment, especially in patients with severe carotid stenosis.

Progesterone improves long-term functional and histological outcomes after permanent stroke in older rats.

Previous studies have shown progesterone to be beneficial in animal models of central nervous system injury, but less is known about its longer-term sustained effects on recovery of function following stroke. We evaluated progesterones effects on a panel of behavioral tests up to 8 weeks after permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats 12m.o. were subjected to pMCAO and, beginning 3h post-pMCAO, given intraperitoneal injections of progesterone (8mg/kg) or vehicle, followed by subcutaneous injections at 8h and then every 24h for 7 days, with tapering of the last 2 treatments. The rats were then tested on functional recovery at 3, 6 and 8 weeks post-stroke. We observed that progesterone-treated animals showed attenuation of infarct volume and improved functional outcomes at 8 weeks after stroke on grip strength, sensory neglect, motor coordination and spatial navigation tests. Progesterone treatments significantly improved motor deficits in the affected limb on a number of gait parameters. Glial fibrillary acidic protein expression was increased in the vehicle group and considerably lowered in the progesterone group at 8 weeks post-stroke. With repeated post-stroke testing, sensory neglect and some aspects of spatial learning performance showed spontaneous recovery, but on gait and grip-strength measres progesterone given only in the acute stage of stroke (first 7 days) showed sustained beneficial effects on all other measures of functional recovery up to 8 weeks post-stroke.

Prehospital Intubation: Further Confounders in Trial Results

Dear Editor, In “Prehospital Intubation is Associated with Favorable Outcomes and Lower Mortality in ProTECT III,” Denninghoff et al. (1) evaluated the relationship between prehospital intubation, functional outcomes, and mortality using data from the patients enrolled in the ProTECT III multicenter, randomized, doubleblind, placebo-controlled trial of early administration of progesterone after traumatic brain injury (2). The authors report that prehospital intubation and air medical transport together were associated with much more favorable outcomes and lower mortality. This observation is interesting and potentially very important for improving emergency services in the field, but their findings also raise concerns regarding how the factors examined in this study may have modified the outcomes of the ProTECT III trial.

Intralipid Vehicle Does Not Interfere with the Efficacy of Progesterone in Attenuating Edema following Traumatic Brain Injury

Abstract The recent disappointing results of phase III trials for progesterone (PROG) in traumatic brain injury (TBI) have triggered speculation about reasons for the negative outcomes. One confounding factor may have been the vehicle used to administer PROG. Virtually all of the many pre-clinical experiments informing the clinical trials and reporting beneficial PROG effects used more soluble 2-hydroxypropyl-b-cyclodextrin as a vehicle given intraperitoneally or subcutaneously rather than a lipid formulation given intravenously (IV). The present investigation compared the effect of PROG infusion with that of lipid emulsion (Intralipid®) as a carrier/vehicle on edema following TBI in rats. Eight-mg/kg doses of PROG with 20% Intralipid were given IV via central venous catheter beginning 1 h post-injury over a 1 h duration (1.2 mL/h). Animals were killed and brains removed at 24 h post-injury. All the brain-injured groups showed more edema compared with the control group. However, PROG+Intralipid significan...The recent disappointing results of phase III trials for progesterone (PROG) in traumatic brain injury (TBI) have triggered speculation about reasons for the negative outcomes. One confounding factor may have been the vehicle used to administer PROG. Virtually all of the many pre-clinical experiments informing the clinical trials and reporting beneficial PROG effects used more soluble 2-hydroxypropyl-b-cyclodextrin as a vehicle given intraperitoneally or subcutaneously rather than a lipid formulation given intravenously (IV). The present investigation compared the effect of PROG infusion with that of lipid emulsion (Intralipid®) as a carrier/vehicle on edema following TBI in rats. Eight-mg/kg doses of PROG with 20% Intralipid were given IV via central venous catheter beginning 1 h post-injury over a 1 h duration (1.2 mL/h). Animals were killed and brains removed at 24 h post-injury. All the brain-injured groups showed more edema compared with the control group. However, PROG+Intralipid significantly attenuated cerebral swelling compared with Intralipid alone. No difference was observed between the TBI-alone and Intralipid groups. Although this study used much a smaller volume and shorter duration of Intralipid infusion than the clinical trials (up to 5 days of continuous infusion), our results suggest that the use of Intralipid in rats did not prevent or mask the beneficial effect of PROG.

Development of a novel progesterone analog in the treatment of traumatic brain injury

ABSTRACT Although systemic progesterone (PROG) treatment has been shown to be neuroprotective by many laboratories and in multiple animal models of brain injury including traumatic brain injury (TBI), PROGs poor aqueous solubility limits its potential for use as a therapeutic agent. The problem of solubility presents challenges for an acute intervention for neural injury, when getting a neuroprotectant to the brain quickly is crucial. Native PROG (nPROG) is hydrophobic and does not readily dissolve in an aqueous‐based medium, so this makes it harder to give under emergency field conditions. An agent with properties similar to those of PROG but easier to store, transport, formulate, and administer early in emergency trauma situations could lead to better and more consistent clinical outcomes following TBI. At the same time, the engineering of a new molecule designed to treat a complex systemic injury must anticipate a range of translational issues including solubility and bioavailability. Here we describe the development of EIDD‐1723, a novel, highly stable PROG analog with >104‐fold higher aqueous solubility than that of nPROG. We think that, with further testing, EIDD‐1723 could become an attractive candidate use as a field‐ready treatment for TBI patients. This article is part of the Special Issue entitled “Novel Treatments for Traumatic Brain Injury”. HIGHLIGHTSEIDD‐1723 is a more water‐soluble, easier‐to‐administer analog of progesterone.Its active metabolite binds to the progesterone receptor with high affinity.EIDD‐1723 shows neuroprotective effects comparable to progesterones.EIDD‐1723 offers new potential as a clinical treatment for TBI.

Effect of Progesterone on Cerebral Vasospasm and Neurobehavioral Outcomes in a Rodent Model of Subarachnoid Hemorrhage

BACKGROUND Subarachnoid hemorrhage (SAH) induces widespread inflammation leading to cellular injury, vasospasm, and ischemia. Evidence suggests that progesterone (PROG) can improve functional recovery in acute brain injury owing to its anti-inflammatory and neuroprotective properties, which could also be beneficial in SAH. We hypothesized that PROG treatment attenuates inflammation-mediated cerebral vasospasm and microglial activation, improves synaptic connectivity, and ameliorates functional recovery after SAH. METHODS We investigated the effect of PROG in a cisternal SAH model in adult male C57BL/6 mice. Neurobehavioral outcomes were evaluated using rotarod latency and grip strength tests. Basilar artery perimeter, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor 1 (GluR1)/synaptophysin colocalization, and Iba-1 immunoreactivity were quantified histologically. RESULTS PROG (8 mg/kg) significantly improved rotarod latency at day 6 and grip strength at day 9. PROG-treated mice had significantly reduced basilar artery vasospasm at 24 hours. GluR1/synaptophysin colocalization, indicative of synaptic GluR1, was significantly reduced in the SAH+Vehicle group at 24 hours, and PROG treatment significantly attenuated this reduction. PROG treatment significantly reduced microglial cell activation and proliferation in cerebellum and cortex but not in the brainstem at 10 days. CONCLUSIONS PROG treatment ameliorated cerebral vasospasm, reduced microglial activation, restored synaptic GluR1 localization, and improved neurobehavioral performance in a murine model of SAH. These results provide a rationale for further translational testing of PROG therapy in SAH.