Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters.

OBJECTIVE The application of pharmacological therapeutics in neurological disorders is limited by the ability of these agents to penetrate the blood-brain barrier (BBB). Focused ultrasound (FUS) has recently gained attention for its potential application as a method for locally opening the BBB and thereby facilitating drug delivery into the brain parenchyma. However, this method still requires optimization to maximize its safety and efficacy for clinical use. In the present study, the authors examined several sonication parameters of FUS influencing BBB opening in small animals. METHODS Changes in BBB permeability were observed during transcranial sonication using low-intensity FUS in 20 adult male Sprague-Dawley rats. The authors examined the effects of FUS sonication with different sonication parameters, varying acoustic pressure, center frequency, burst duration, microbubble (MB) type, MB dose, pulse repetition frequency (PRF), and total exposure time. The focal region of BBB opening was identified by Evans blue dye. Additionally, H & E staining was used to identify blood vessel damage. RESULTS Acoustic pressure amplitude and burst duration were closely associated with enhancement of BBB opening efficiency, but these parameters were also highly correlated with tissue damage in the sonicated region. In contrast, MB types, MB dose, total exposure time, and PRF had an influence on BBB opening without conspicuous tissue damage after FUS sonication. CONCLUSIONS The study aimed to identify these influential conditions and provide safety and efficacy values for further studies. Future work based on the current results is anticipated to facilitate the implementation of FUS sonication for drug delivery in various CNS disease states in the near future.

Urokinase, a promising candidate for fibrinolytic therapy for intracerebral hemorrhage.

OBJECTIVE Intracerebral hemorrhage (ICH) is associated with a high rate of mortality and severe disability, while fibrinolysis for ICH evacuation is a possible treatment. However, reported adverse effects can counteract the benefits of fibrinolysis and limit the use of tissue-type plasminogen activator (tPA). Identifying appropriate fibrinolytics is still needed. Therefore, the authors here compared the use of urokinase-type plasminogen activator (uPA), an alternate thrombolytic, with that of tPA in a preclinical study. METHODS Intracerebral hemorrhage was induced in adult male Sprague-Dawley rats by injecting autologous blood into the caudate, followed by intraclot fibrinolysis without drainage. Rats were randomized to receive uPA, tPA, or saline within the clot. Hematoma and perihematomal edema, brain water content, Evans blue fluorescence and neurological scores, matrix metalloproteinases (MMPs), MMP mRNA, blood-brain barrier (BBB) tight junction proteins, and nuclear factor-κB (NF-κB) activation were measured to evaluate the effects of these 2 drugs in ICH. RESULTS In comparison with tPA, uPA better ameliorated brain edema and promoted an improved outcome after ICH. In addition, uPA therapy more effectively upregulated BBB tight junction protein expression, which was partly attributed to the different effects of uPA and tPA on the regulation of MMPs and its related mRNA expression following ICH. CONCLUSIONS This study provided evidence supporting the use of uPA for fibrinolytic therapy after ICH. Large animal experiments and clinical trials are required to further explore the efficacy and safety of uPA in ICH fibrinolysis.

Structural and biochemical abnormalities in the absence of acute deficits in mild primary blast-induced head trauma.

OBJECTIVE: Blast-induced neurotrauma (BINT), if not fatal, is nonetheless potentially crippling. It can produce a wide array of acute symptoms in moderate-to-severe exposures, but mild BINT (mBINT) is characterized by the distinct absence of acute clinical abnormalities. The lack of observable indications for mBINT is particularly alarming, as these injuries have been linked to severe long-term psychiatric and degenerative neurological dysfunction. Although the long-term sequelae of BINT are extensively documented, the underlying mechanisms of injury remain poorly understood, impeding the development of diagnostic and treatment strategies. The primary goal of this research was to recapitulate primary mBINT in rodents in order to facilitate well-controlled, long-term investigations of blast-induced pathological neurological sequelae and identify potential mechanisms by which ongoing damage may occur postinjury. METHODS: A validated, open-ended shock tube model was used to deliver blast overpressure (150 kPa) to anesthetized rats with body shielding and head fixation, simulating the protective effects of military-grade body armor and isolating a shock wave injury from confounding systemic injury responses, head acceleration, and other elements of explosive events. Evans Blue-labeled albumin was used to visualize blood-brain barrier (BBB) compromise at 4 hours postinjury. Iba1 staining was used to visualize activated microglia and infiltrating macrophages in areas of peak BBB compromise. Acrolein, a potent posttraumatic neurotoxin, was quantified in brain tissue by immunoblotting and in urine through liquid chromatography with tandem mass spectrometry at 1, 2, 3, and 5 days postinjury. Locomotor behavior, motor performance, and short-term memory were assessed with open field, rotarod, and novel object recognition (NOR) paradigms at 24 and 48 hours after the blast. RESULTS: Average speed, maximum speed, and distance traveled in an open-field exploration paradigm did not show significant differences in performance between sham-injured and mBINT rats. Likewise, rats with mBINT did not exhibit deficits in maximum revolutions per minute or total run time in a rotarod paradigm. Short-term memory was also unaffected by mBINT in an NOR paradigm. Despite lacking observable motor or cognitive deficits in the acute term, blast-injured rats displayed brain acrolein levels that were significantly elevated for at least 5 days, and acrolein's glutathione-reduced metabolite, 3-HPMA, was present in urine for 2 days after injury. Additionally, mBINT brain tissue demonstrated BBB damage 4 hours postinjury and colocalized neuroinflammatory changes 24 hours postinjury. CONCLUSIONS: This model highlights mBINT's potential for underlying detrimental physical and biochemical alterations despite the lack of apparent acute symptoms and, by recapitulating the human condition, represents an avenue for further examining the pathophysiology of mBINT. The sustained upregulation of acrolein for days after injury suggests that acrolein may be an upstream player potentiating ongoing postinjury damage and neuroinflammation. Ultimately, continued research with this model may lead to diagnostic and treatment mechanisms capable of preventing or reducing the severity of long-term neurological dysfunction following mBINT.

Focal cerebral ischemic tolerance and change in blood-brain barrier permeability after repetitive pure oxygen exposure preconditioning in a rodent model.

OBJECTIVE The goal of this study was to demonstrate that repetitive pure oxygen exposure preconditioning (O2PC) for 8 hours per day for 3 or 7 days, a practicable preconditioning for clinical use, is able to induce cerebral ischemic tolerance (IT) and further clarify the accompanying changes in the blood-brain barrier (BBB) that may be involved. METHODS A total of 68 adult male Sprague-Dawley rats and eight 1-day-old rat pups were used in this study. The adult rats were exposed to pure O2 (38 rats) 8 hours a day for 3 or 7 days or to room air (in an identical setup) for 8 hours a day for 7 days as controls (30 rats). Arterial O2 tension (PaO2) was measured in 6 rats exposed to O2 and 3 controls. Focal cerebral ischemia was elicited by middle cerebral artery occlusion (MCAO) in 37 rats, of which 21 had been exposed to pure O2 for 3 or 7 days and 16 to room air for 7 days as controls. Neurological behavior was scored with the Garcia score in 15 MCAO rats, of which 10 had been exposed to pure O2 for 3 or 7 days and 5 to room air for 7 days as controls, and cerebral infarct volumes were assessed with TTC (2,3,5-triphenyltetrazolium chloride) staining in 10 rats (5 from each group) after 7 days of exposure. Formamide-extraction method was used to detect leakage of Evans blue (EB) dye in 7 rats exposed to pure O2 for 7 days and 7 exposed to room air for 7 days. Fluorescence microscopy was used to analyze the leaked EB in the nonischemic areas of 4 rats exposed to pure O2 for 7 days and 4 exposed to room air for 7 days before MCAO and the brain of the rats that had not been subjected to MCAO. Astrocyte changes associated with O2PC were evaluated by means of fluorescence microscopy and electron microscopy in 14 rats that were exposed to the same O2 or control conditions as the MCAO rats but without MCAO. Astrocytes were also obtained from 8 rat pups and cultured; levels of AQP4 and VEGF were detected by Western blot and ELISA in cells with and without O2 treatment. RESULTS A significant increase in PaO2 was seen after O2PC. The neurological score was significantly increased in the O2PC groups (10.6 ± 0.6 in the 3-day O2PC group, p < 0.05; 12 ± 0.84 in the 7-day O2PC group, p < 0.05) compared with the control group (7 ± 0.55). The ratio of cerebral infarct volume to contralateral cerebral hemisphere volume was significantly lower in the O2PC group than in the control group (0.204 ± 0.03 vs 0.48 ± 0.05, p < 0.05). The amount of leaked EB in the ischemic cerebral hemisphere was also lower in the O2-treated rats than in controls (7.53 ± 1.4 vs 11.79 ± 3.3 µg EB/g brain weight, p < 0.05). However, fluorescence microscopy showed significantly greater BBB permeability in the nonischemic areas in the O2PC group than in controls (p < 0.05). More red fluorescence could be observed in the nonischemic areas in both the ipsilateral and contralateral sides of the ischemic brain in the O2PC animals than in the nonischemic areas in the corresponding sides of the controls. Further investigation of the effect of the O2PC itself on the BBB of rats that were not subjected to MCAO showed that there was no EB leakage in the brain parenchyma in the rats exposed to room air, but some red fluorescence patches were noticed in the normal brain from the rats in the O2PC group. Astrocytes, including those from areas around the BBB, were activated in the O2PC group. Levels of both aquaporin 4 (AQP4) and vascular endothelial growth factor (VEGF) were significantly increased in cultured astrocytes after O2PC. CONCLUSIONS These findings suggest that O2PC is able to induce IT, which makes it a strong candidate for clinical use. Moreover, O2PC can also promote BBB opening, which may contribute to the induction of IT as well as representing a possible strategy for promoting drug transportation into the CNS. Activated astrocytes are likely to be involved in these processes through astrocyte-derived factors, such as AQP4 and VEGF.

Amelioration of oxidative stress and protection against early brain injury by astaxanthin after experimental subarachnoid hemorrhage.

UNLABELLED: OBJECT.: Aneurysmal subarachnoid hemorrhage (SAH) causes devastating rates of mortality and morbidity. Accumulating studies indicate that early brain injury (EBI) greatly contributes to poor outcomes after SAH and that oxidative stress plays an important role in the development of EBI following SAH. Astaxanthin (ATX), one of the most common carotenoids, has a powerful antioxidative property. However, the potential role of ATX in protecting against EBI after SAH remains obscure. The goal of this study was to assess whether ATX can attenuate SAH-induced brain edema, blood-brain barrier permeability, neural cell death, and neurological deficits, and to elucidate whether the mechanisms of ATX against EBI are related to its powerful antioxidant property. METHODS: Two experimental SAH models were established, including a prechiasmatic cistern SAH model in rats and a one-hemorrhage SAH model in rabbits. Both intracerebroventricular injection and oral administration of ATX were evaluated in this experiment. Posttreatment assessments included neurological scores, body weight loss, brain edema, Evans blue extravasation, Western blot analysis, histopathological study, and biochemical estimation. RESULTS: It was observed that an ATX intracerebroventricular injection 30 minutes post-SAH could significantly attenuate EBI (including brain edema, blood-brain barrier disruption, neural cell apoptosis, and neurological dysfunction) after SAH in rats. Meanwhile, delayed treatment with ATX 3 hours post-SAH by oral administration was also neuroprotective in both rats and rabbits. In addition, the authors found that ATX treatment could prevent oxidative damage and upregulate the endogenous antioxidant levels in the rat cerebral cortex following SAH. CONCLUSIONS: These results suggest that ATX administration could alleviate EBI after SAH, potentially through its powerful antioxidant property. The authors conclude that ATX might be a promising therapeutic agent for EBI following SAH.