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Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (− 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of − 3.0 °C.

Peripheral Neuropathies (PN) are common conditions whose treatment is still lacking in most cases. Animal models are crucial, but experimental procedures should be refined in some cases. We performed a detailed characterization of the ventral caudal nerve to contribute to a more effective assessment of axonal damage in future PN studies. PN was induced via weekly systemic injection of a neurotoxic drug (paclitaxel); we compared the control and PN-affected rats, performing serial neurophysiological evaluations of the caudal nerve for its entire length. On the same nerve portions, we performed light microscopy and ultrastructural pathological observations to assess the severity of damage and verify the integrity of the surrounding structures. Neurophysiological and morphological analyses confirmed that a severe axonopathy had ensued in the PN group, with a length-dependent modality, matching morphological observations. The site of neurophysiological recording (e.g., distance from the base of the tail) was critical for achieving useful data. A flexible experimental paradigm should be considered in animal studies investigating axonal PN, particularly if the expected severity is relevant; the mid-portion of the tail might be the most appropriate site: there damage might be remarkable but neither as extreme as at the tip of the tail nor as mild as at the base of the tail.

Neurological outcome after ischemic stroke depends on residual salvageable brain tissue at the time of recanalization. Head down tilt 15° (HDT15) was proven effective in reducing infarct size and improving functional outcome in rats with transient middle cerebral artery occlusion (t-MCAO) by increasing cerebral perfusion within the ischemic penumbra. In this pooled analysis, individual animal-level data from three experimental series were combined in a study population of 104 t-MCAO rats (45 in HDT15 group and 59 in flat position group). Co-primary outcomes were infarct size and functional outcome at 24 h in both groups. The secondary outcome was hemodynamic change induced by HDT15 in ischemic and non-ischemic hemispheres in a subgroup of animals. Infarct size at 24 h was smaller in HDT15 group than in flat position group (absolute mean difference 31.69 mm

Glioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time- and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.

Chemotherapy-Induced Peripheral Neurotoxicity (CIPN) is a severe and long-lasting side effect of anticancer therapy, which can severely impair patients’ quality of life. It is a sensory and length-dependent neuropathy, which predominantly affects large myelinated fibers. Easy and reliable monitoring of CIPN in patients is still an unmet clinical need. Since increasing clinical evidence supports the potential use of neurofilament light chain (NfL) as a biomarker of axonal injury, in this study we measured serum NfL levels in animals chronically treated with cisplatin (CDDP) and paclitaxel (PTX), two antineoplastic drugs with different neuronal targets. Wistar rats were treated with CDDP (2 mg/kg i.p. twice/week for 4 weeks) or PTX (10 mg/kg i.v. once/week for 4 weeks). Repeated serum NfL quantification was obtained using the Single Molecule Array (Simoa) technology. The onset and progression of peripheral neurotoxicity were evaluated through neurophysiology, morphological assessments and intraepidermal nerve fibers density quantification. Our results showed that serum NfL measurements correlated with the severity of axonal damage. In fact, both treatments induced serum NfL increase, but higher levels were evidenced in PTX-treated animals, compared with CDDP-treated rats, affected by a milder neurotoxicity. Notably, also the timing of the NfL level increase was associated with the severity of morphological and functional alterations of axonal structure. Therefore, NfL could be a useful biomarker for axonal damage in order to follow the onset and severity of axonal degeneration and possibly limit the occurrence of serious PNS disease.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

Proceedings of the 31st National Conference of the Italian Group for the Study of Neuromorphology “Gruppo Italiano per lo Studio della Neuromorfologia” G.I.S.N., Milan, November 26-27, 2021

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Background Peripheral neuropathy treatment is not always satisfactory. To fill this gap, inferences from bench side are warranted, where morphological and pathogenetic determinations can be performed. Nerve conduction studies (NCS) are ideal to translate results from preclinical to clinical setting. New methods We propose a comprehensive 8-minute protocol for sensory-motor neurophysiological assessment, similar to routine clinical practice: sensory proximal and distal caudal nerves, motor caudal nerve, and sensory digital nerve recordings were used and tested in 2 different experimental settings. In Experiment 1 we compared control (CTRL) animals to a severe sensory-motor polyneuropathy (animals treated with vincristine [VCR]), and in Experiment 2 CTRL animals were compared to a mild sensory polyneuropathy (animals treated with oxaliplatin [OHP]). NCS were performed after 1-month of chemotherapy and matched with confirmatory neuropathological analyses. Results VCR treated animals showed, at NCS, a relevant sensory-motor polyneuropathy ensued at the end of treatment; whereas, OHP animals showed a mild distal sensory neuropathy. These patterns were confirmed by neuropathological analysis. Comparison with existing methods In literature, the majority of proposed neurophysiological protocols relies mainly on a single nerve testing, rather than a combination of them, and only a few studies tested both caudal and sciatic nerve branches, nevertheless not aiming at fully reproduce clinical protocols (e.g., seeking for length-dependency); to provide evidence of appropriateness of our protocol we applied a gold standard: neuropathology. Conclusion The simple and rapid protocol here presented can be suggested as a good translation outcome measure in preclinical setting.

BACKGROUND: Head down tilt 15° (HDT15°), applied before recanalization, increases collateral flow and improves outcome in experimental ischemic stroke. For its simplicity and low cost, HDT15° holds considerable potential to be developed as an emergency treatment of acute stroke in the pre-hospital setting, where hemorrhagic stroke is the major mimic of ischemic stroke. In this study, we assessed safety of HDT15° in the acute phase of experimental intracerebral hemorrhage. METHODS: Intracerebral hemorrhage was produced by stereotaxic injection of collagenase in Wistar rats. A randomized non-inferiority trial design was used to assign rats to HDT15° or flat position (n=64). HDT15° was applied for 1 hour during the time window of hematoma expansion. The primary outcome was hematoma volume at 24 hours. Secondary outcomes were mass effect, mortality and functional deficit in the main study and acute changes of intracranial pressure, hematoma growth and cardiorespiratory parameters in separate sets of randomized animals (n=32). RESULTS: HDT15° achieved the specified criteria of non-inferiority for hematoma volume at 24 hours. Mass effect, mortality and functional deficit at 24 hours showed no difference in the two groups. HDT15° induced a mild increase in intracranial pressure with respect to the pre-treatment values (+ 2.91 +/- 1.76 mmHg). HDT15° had a neutral effect on MRI-based analysis of hematoma growth and cardiorespiratory parameters. CONCLUSIONS: Application of HDT15° in the hyperacute phase of experimental intracerebral hemorrhage does not worsen early outcome. Further research is needed to implement HDT15° as an emergency collateral therapeutic for acute stroke.

Oxaliplatin-induced peripheral neuropathy is characterized by an acute hyperexcitability syndrome triggered/exacerbated by cold. The mechanisms underlying oxaliplatin-induced peripheral neuropathy are unclear, but the alteration of ion channel expression and activity plays a well-recognized central role. Recently, we found that oxaliplatin leads to cytosolic acidification in dorsal root ganglion (DRG) neurons. Here, we investigated the early impact of oxaliplatin on the proton-sensitive TREK potassium channels. Following a 6-h oxaliplatin treatment, both channels underwent a transcription upregulation that returned to control levels after 42 h. The overexpression of TREK channels was also observed after in vivo treatment in DRG cells from mice exposed to acute treatment with oxaliplatin. Moreover, both intracellular pH and TREK channel transcription were similarly regulated after incubation with amiloride, an inhibitor of the Na+/H+ exchanger. In addition, we studied the role of oxaliplatin-induced acidification on channel behavior, and, as expected, we observed a robust positive modulation of TREK channel activity. Finally, we focused on the impact of this complex modulation on capsaicin-evoked neuronal activity finding a transient decrease in the average firing rate following 6 h of oxaliplatin treatment. In conclusion, the early activation of TREK genes may represent a mechanism of protection against the oxaliplatin-related perturbation of neuronal excitability.

Oxaliplatin (OHP) is an antineoplastic compound able to induce peripheral neurotoxicity. Oxidative stress has been suggested to be a key factor in the development of OHP-related peripheral neurotoxicity. Mangafodipir, a contrast agent possessing mitochondrial superoxide dismutase (MnSOD)-mimetic activity, has been tested as a cytoprotector in chemotherapy-induced peripheral neurotoxicity (CIPN). Calmangafodipir (PledOx&reg, ) has even better therapeutic activity. We investigated a BALB/c mouse model of OHP-related CIPN and the effects of the pre-treatment of calmangafodipir (2.5, 5, or 10 mg/kg intravenously) on sensory perception, and we performed a pathological study on skin biopsies to assess intraepidermal nerve fiber (IENF) density. At the end of the treatments, OHP alone or in pre-treatment with calmangafodipir 2.5 and 10 mg/kg, induced mechanical allodynia and cold thermal hyperalgesia, but calmangafodipir 5 mg/kg prevented these effects. Accordingly, OHP alone or in pre-treatment with calmangafodipir 2.5 and 10 mg/kg, induced a significant reduction in IENF density, but calmangafodipir 5 mg/kg prevented this reduction. These results confirm a protective effect of calmangafodipir against OHP-induced small fiber neuropathy. Interestingly, these results are in agreement with previous observations suggesting a U-shaped effect of calmangafodipir, with the 10 mg/kg dose less effective than the lower doses.

One of the most relevant dose-limiting adverse effects of platinum drugs is the development of a sensory peripheral neuropathy that highly impairs the patients' quality of life. Nowadays there are no available efficacy strategies for the treatment of platinum-induced peripheral neurotoxicity (PIPN), and the only way to prevent its development and progression is by reducing the dose of the cytostatic drug or even withdrawing the chemotherapy regimen. This clinical issue has been the main focus of hundreds of preclinical research works during recent decades. As a consequence, dozens of in vitro and in vivo models of PIPN have been developed to elucidate the molecular mechanisms involved in its development and to find neuroprotective targets. The apoptosis of peripheral neurons has been identified as the main mechanism involved in PIPN pathogenesis. This mechanism of DRG sensory neurons cell death is triggered by the nuclear and mitochondrial DNA platination together with the increase of the oxidative cellular status induced by the depletion of cytoplasmic antioxidant mechanisms. However, since there has been no successful transfer of preclinical results to clinical practise in terms of therapeutic approaches, some mechanisms of PIPN pathogenesis still remain to be elucidated. This review is focused on the pathogenic mechanisms underlying PIPN described up to now, provided by the critical analysis of in vitro and in vivo models.

Chemotherapy-induced peripheral neurotoxicity represents one of the most relevant dose-limiting side effects that can affect cancer patients treated with the common antineoplastic agents. Since the severity of neurotoxicity often leads to dose reduction or early cessation of chemotherapy, the investigation of molecular mechanisms underlying chemotherapy-induced peripheral neurotoxicity is an urgent clinical need in order to better understand its physiopathology and find effective strategies for neuroprotection. Several in vivo preclinical models of chemotherapy-induced peripheral neurotoxicity have been developed but a great variability in mouse strain, dose, route of administration of the drug, treatment schedule and assessment of neurotoxicity is observed between the different published studies making difficult the comparison and interpretation of their results. In many of these studies only behavioural tests are used as outcome measures, while possible neurophysiological and neuropathological changes are not evaluated. In this study, focused on experimental oxaliplatin-induced peripheral neurotoxicity, we reproduced and compared four mouse models with very different drug dose (low or high dose-intensity) and treatment schedules (short or long-term treatment), selected from the literature. Using a multimodal assessment based on behavioural, neurophysiological and neuropathological methods, we evidenced remarkable differences in the results obtained in the selected animal models. This work suggests the importance of a multimodal approach including extensive pathological investigation to confirm the behavioural results.

Cerebral collaterals are dynamically recruited after arterial occlusion and highly affect tissue outcome in acute ischemic stroke. We investigated the efficacy and safety of four pathophysiologically distinct strategies for acute modulation of collateral flow (collateral therapeutics) in the rat stroke model of transient middle cerebral artery (MCA) occlusion. A composed randomization design was used to assign rats (n = 118) to receive phenylephrine (induced hypertension), polygeline (intravascular volume load), acetazolamide (cerebral arteriolar vasodilation), head down tilt (HDT) 15° (cerebral blood flow diversion), or no treatment, starting 30 min after MCA occlusion. Compared to untreated animals, treatment with collateral therapeutics was associated with lower infarct volumes (62% relative mean difference; 51.57 mm3 absolute mean difference; p

Oxaliplatin (OHP) Induced Peripheral Neurotoxicity (OIPN) is one of the dose-limiting toxicities of the drug and these adverse effects limit cancer therapy with L-OHP, used for colorectal cancer treatment. Acute neurotoxicity consists of symptoms that are the hallmarks of a transient axonal hyperexcitability; chronic neurotoxicity has a clinical picture compatible with a length-dependent sensory neuropathy. Acute OIPN pathogenesis has been linked to sodium voltage-operated channels (Na + VOC) dysfunction and it has been advocated as a possible predisposing factor to chronic neurotoxicity. We tested if topiramate (TPM), a well-known Na + VOC modulator, was able to modify acute as well as chronic OIPN. The project was divided into two parts. In Experiment 1 we tested by means of Nerve Excitability Testing (NET) a cohort of female Wistar rats to assess TPM effects after a single OHP administration (5 mg/kg, iv). In Experiment 2 we assessed TPM effects after chronic OHP treatment (5 mg/kg, 2qw4ws, iv) using NET, nerve conduction studies (NCS), behavioral tests and neuropathology (caudal nerve morphometry and morphology and Intraepidermal Nerve Fiber [IENF] density). In Experiment 1 TPM was able to prevent OHP effects on Na + VOC: OHP treatment induced a highly significant reduction of the sensory nerve's threshold, during the superexcitability period (p-value = 0.008), whereas TPM co-administration prevented this effect. In Experiment 2 we verified that TPM was able to prevent not only acute phenomena, but also to completely prevent chronic OIPN. This latter observation was supported by a multimodal approach: in fact, only OHP group showed altered findings compared to CTRL group at a neurophysiological (proximal caudal nerve sensory nerve action potential [SNAP] amplitude, p-value = 0.001; distal caudal nerve SNAP amplitude, p-value

Chemotherapy-Induced Peripheral Neurotoxicity (CIPN) is often dose-limiting and impacts life quality and survival of cancer patients. Ghrelin agonists have neuroprotectant effects and may have a role in treating or preventing CIPN. We evaluated the CNS-penetrant ghrelin agonist HM01 in three experimental models of CIPN at doses of 3–30 mg/kg p.o. daily monitoring orexigenic properties, nerve conduction, mechanical allodynia, and intra-epidermal nerve fiber density (IENFD). In a cisplatin-based study, rats were dosed daily for 3 days (0.5 mg/kg i.p.) + HM01. Cisplatin treatment induced mechanical hypersensitivity which was significantly reduced by HM01. In a second study, oxaliplatin was administered to mice (6 mg/kg i.p. 3 times/week for 4 weeks) resulting in significant digital nerve conduction velocity (NCV) deficits and reduction of IENFD. Concurrent HM01 dose dependently prevented the decline in NCV and attenuated the reduction in IENFD. Pharmacokinetic studies showed HM01 accumulation in the dorsal root ganglia and sciatic nerves which reached concentrations > 10 fold that of plasma. In a third model, HM01 was tested in preventive and therapeutic paradigms in a bortezomib-based rat model (0.2 mg/kg i.v., 3 times/week for 8 weeks). In the preventive setting, HM01 blocked bortezomib-induced hyperalgesia and IENFD reduction at all doses tested. In the therapeutic setting, significant effect was observed, but only at the highest dose. Altogether, the robust peripheral nervous system penetration of HM01 and its ability to improve multiple oxaliplatin-, cisplatin-, and bortezomib-induced neurotoxicities suggest that HM01 may be a useful neuroprotective adjuvant for CIPN.

Background Chemotherapy-induced peripheral neurotoxicity (CIPN) is a severe adverse effect in patients receiving antitumor agents, and no effective treatment is available. Although the mechanisms responsible for the development of CIPN are poorly understood, recent findings make neuroinflammation an attractive target to be investigated, particularly when neuropathic pain is a prominent feature such as after bortezomib administration. The aim of our study was to evaluate the effect of intravenous immunoglobulins (IVIg) delivery in chronic CIPN. The related neuro-immune aspects were investigated in a well-characterized rat model of bortezomib-induced peripheral neurotoxicity (BIPN). Methods After determination of a suitable schedule based on a preliminary pharmacokinetic pilot study, female Wistar rats were treated with IVIg 1 g/kg every 2 weeks. IVIg treatment was started at the beginning of bortezomib administration (“preventive” schedule), or once BIPN was already ensued after 4 weeks of treatment (“therapeutic” schedule). Neurophysiological and behavioral studies were performed to assess the extent of painful peripheral neurotoxicity induced by bortezomib, and these functional assessments were completed by pathologic examination of peripheral nerves and intraepidermal nerve fiber quantification (IENF). The role of the innate immune response in BIPN was investigated by immunochemistry characterization of macrophage infiltration in peripheral nerves. Results Both schedules of IVIg administration were able to significantly reduce bortezomib-induced heat and mechanical allodynia. Although these changes were not evidenced at the neurophysiological examination of peripheral nerves, they behavioral effects were paralleled in the animals treated with the preventive schedule by reduced axonopathy in peripheral nerves and significant protection from loss of IENF. Moreover, IVIg administration was very effective in reducing infiltration in peripheral nerves of macrophages with the M1, pro-inflammatory phenotype. Conclusion Our results suggest a prominent role of neuroinflammation in BIPN and that IVIg might be considered as a possible safe and effective therapeutic option preventing M1 macrophage infiltration. However, since neuropathic pain is frequent also in other CIPN types, it also indicates the need for further investigation in other forms of CIPN.

The objective of this study is to test the feasibility of using serum neurofilament light chain (NfL) as a disease biomarker in Chemotherapy Induced Peripheral Neuropathy (CIPN) since this easy accessible biological test may have a large impact on clinical management and safety of cancer patients. We performed this preclinical study using a well-characterized rat model based on repeated administration of the cytostatic drug vincristine (VCR, 0.2 mg/kg intravenously via the tail vein once/week for 4 times). Serial NfL serum concentration was measured using the in-house Simoa NfL assay and peripheral neuropathy onset was measured by sensory and motor nerve conduction studies. Serum NfL measure in untreated and VCR-treated rats demonstrated a steady, and significant increase during the course of VCR administration, with a final 4-fold increase with respect to controls (p

International audience; High variability in infarct size is common in experimental stroke models and affects statistical power and validity of neuroprotection trials. The aim of this study was to explore cerebral collateral flow as a stratification factor for the prediction of ischemic outcome. Transient intraluminal occlusion of the middle cerebral artery was induced for 90 min in 18 Wistar rats. Cerebral collateral flow was assessed intra-procedurally using multi-site laser Doppler flowmetry monitoring in both the lateral middle cerebral artery territory and the borderzone territory between middle cerebral artery and anterior cerebral artery. Multi-modal magnetic resonance imaging was used to assess acute ischemic lesion (diffu-sion-weighted imaging, DWI), acute perfusion deficit (time-to-peak, TTP), and final ischemic lesion at 24 h. Infarct volumes and typology at 24 h (large hemispheric versus basal ganglia infarcts) were predicted by both intra-ischemic collateral perfusion and acute DWI lesion volume. Collateral flow assessed by multi-site laser Doppler flowmetry correlated with the corresponding acute perfusion deficit using TTP maps. Multi-site laser Doppler flowmetry monitoring was able to predict ischemic outcome and perfusion deficit in good agreement with acute MRI. Our results support the additional value of cerebral collateral flow monitoring for outcome prediction in experimental ischemic stroke, especially when acute MRI facilities are not available.

International audience; Intracranial collaterals are dynamically recruited after arterial occlusion and are emerging as a strong determinant of tissue outcome in both human and experimental ischemic stroke. The relationship between collateral flow and ischemic penumbra remains largely unexplored in pre-clinical studies. The aim of the present study was to investigate the pattern of collateral flow with regard to penumbral tissue after transient middle cerebral artery (MCA) occlusion in rats. MCA was transiently occluded (90min) by intraluminal filament in adult male Wistar rats (n=25). Intracranial collateral flow was studied in terms of perfusion deficit and biosignal fluctuation analyses using multi-site laser Doppler monitoring. Molecular penumbra was defined by topographical mapping and quantitative signal analysis of Heat Shock Protein 70kDa (HSP70) immunohistochemistry. Functional deficit and infarct volume were assessed 24h after ischemia induction. The results show that functional performance of intracranial collaterals during MCA occlusion inversely correlated with HSP70 immunoreactive areas in both the cortex and the striatum, as well as with infarct size and functional deficit. Intracranial collateral flow was associated with reduced areas of both molecular penumbra and ischemic core and increased areas of intact tissue in rats subjected to MCA occlusion followed by reperfusion. Our findings prompt the development of collateral therapeutics to provide tissue-saving strategies in the hyper-acute phase of ischemic stroke prior to recanalization therapy.

Statins have since long been reported to exert acute neuroprotection in experimental stroke models. However, crucial questions still need to be addressed as far as the timing of their cerebral effects after intravascular administration and the role played by the blood brain barrier (BBB) crossing properties. We tested the effects of an hydrophilic statin (pravastatin, 100 nM), which poorly crosses BBB under physiological conditions. Pravastatin was administered either 90 min before or immediately after transient middle cerebral artery occlusion in the in vitro isolated Guinea pig brain preparation. A multi-modal outcome assessment was performed, through electrophysiological and cerebral vascular tone recordings, MAP-2 immunohistochemistry, BBB evaluation via ZO-1/FITC-albumin analysis, AKT and ERK activation and whole-cell antioxidant capacity. Pravastatin pre-ischemic administration did not produce any significant effect. Pravastatin post-ischemic administration significantly prevented MAP-2 immunoreactivity loss in ischemic areas, increased ERK phosphorylation in the ischemic hemisphere and enhanced whole-cell antioxidant capacity. Electrophysiological parameters, vascular tone and AKT signaling were unchanged. In all tested ischemic brains, ZO-1 fragmentation and FITC albumin extravasation was observed, starting 30 min from ischemia onset, indicating loss of BBB integrity. Our findings indicate that the rapid anti-ischemic effects of intravascular pravastatin are highly dependent on BBB increased permeability after stroke.