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The primary challenge posed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19-related mortality, often exacerbated by additional medical complications, such as COVID-19-associated kidney injuries (CAKIs). Up to half of COVID-19 patients experience kidney complications, with those facing acute respiratory failure and kidney injury having the worst overall prognosis. Despite the significant impact of CAKI on COVID-19-related mortality and its enduring effects in long COVID, the underlying causes and molecular mechanisms of CAKI remain elusive. In this study, we identified a functional relationship between the expression of the SARS-CoV-2 ORF3a protein and inflammation-driven apoptotic death of renal tubular epithelial cells in patients with CAKI. We demonstrate in vitro that ORF3a independently induces renal cell-specific apoptotic cell death, as evidenced by the elevation of kidney injury molecule-1 (KIM-1) and the activation of NF-kB-mediated proinflammatory cytokine (TNFα and IL-6) production. By examining kidney tissues of SARS-CoV-2-infected K18-ACE2 transgenic mice, we observed a similar correlation between ORF3a-induced cytopathic changes and kidney injury. This correlation was further validated through reconstitution of the ORF3a effects via direct adenoviral injection into mouse kidneys. Through medicinal analysis, we identified a natural compound, glycyrrhizin (GL4419), which not only blocks viral replication in renal cells, but also mitigates ORF3a-induced renal cell death by inhibiting activation of a high mobility group box 1 (HMGB1) protein, leading to a reduction of KIM-1. Moreover, ORF3a interacts with HMGB1. Overproduction or downregulation of hmgb1 expression results in correlative changes in renal cellular KIM-1 response and respective cytokine production, implicating a crucial role of HMGB1 in ORF3a-inflicted kidney injuries. Our data suggest a direct functional link between ORF3a and kidney injury, highlighting ORF3a as a unique therapeutic target contributing to CAKI., Importance: The major challenge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the pandemic is COVID-19-related mortality, which has tragically claimed millions of lives. COVID-19-associated morbidity and mortality are often exacerbated by pre-existing medical conditions, such as chronic kidney diseases (CKDs), or the development of acute kidney injury (AKI) due to COVID-19, collectively known as COVID-19-associated kidney injuries (CAKIs). Patients who experience acute respiratory failure with CAKI have the poorest clinical outcomes, including increased mortality. Despite these alarming clinical findings, there is a critical gap in our understanding of the underlying causes of CAKI. Our study establishes a direct correlation between the expression of the SARS-CoV-2 viral ORF3a protein and kidney injury induced by ORF3a linking to CAKI. This functional relationship was initially observed in our clinical studies of COVID-19 patients with AKI and was further validated through animal and in vitro cellular studies, either by expressing ORF3a alone or in the context of viral infection. By elucidating this functional relationship and its underlying mechanistic pathways, our research deepens the understanding of COVID-19-associated kidney diseases and presents potential therapeutic avenues to address the healthcare challenges faced by individuals with underlying conditions., Competing Interests: The authors declare no conflict of interest.

Trauma to the brain can induce a contusion characterized by a discrete intracerebral or diffuse interstitial hemorrhage. In humans, "computed tomography-positive," that is, hemorrhagic, temporal lobe contusions (tlCont) have unique sequelae. TlCont confers significantly increased odds for moderate or worse disability and the inability to return to baseline work capacity compared to intra-axial injuries in other locations. Patients with tlCont are at elevated risks of memory dysfunction, anxiety, and post-traumatic epilepsy due to involvement of neuroanatomical structures unique to the temporal lobe including the amygdala, hippocampus, and ento-/perirhinal cortex. Because of the relative inaccessibility of the temporal lobe in rodents, no preclinical model of tlCont has been described, impeding progress in elucidating the specific pathophysiology unique to tlCont. Here, we present a minimally invasive mouse model of tlCont with the contusion characterized by a traumatic interstitial hemorrhage. Mortality was low and sensorimotor deficits (beam walk, accelerating rotarod) resolved completely within 3-5 days. However, significant deficits in memory (novel object recognition, Morris water maze) and anxiety (elevated plus maze) persisted at 14-35 days and nonconvulsive electroencephalographic seizures and spiking were significantly increased in the hippocampus at 7-21 days. Immunohistochemistry showed widespread astrogliosis and microgliosis, bilateral hippocampal sclerosis, bilateral loss of hippocampal and cortical inhibitory parvalbumin neurons, and evidence of interhemispheric connectional diaschisis involving the fiber bundle in the ventral corpus callosum that connects temporal lobe structures. This model may be useful to advance our understanding of the unique features of tlCont in humans.

Brain ischemia causes disruption in cerebral blood flow and blood-brain barrier (BBB) integrity which are normally maintained by the astrocyte endfeet. Emerging evidence points to dysregulation of the astrocyte translatome during ischemia, but its effects on the endfoot translatome are unknown. In this study, we aimed to investigate the early effects of ischemia on the astrocyte endfoot translatome in a rodent model of cerebral ischemia-reperfusion. To do so, we immunoprecipitated astrocyte-specific tagged ribosomes (RiboTag IP) from mechanically isolated brain microvessels. In mice subjected to middle cerebral artery occlusion and reperfusion and contralateral controls, we sequenced ribosome-bound RNAs from perivascular astrocyte endfeet and identified 205 genes that were differentially expressed in the translatome after ischemia. Pathways associated with the differential expressions included proteostasis, inflammation, cell cycle, and metabolism. Transcription factors whose targets were enriched amongst upregulated translating genes included HSF1, the master regulator of the heat shock response. The most highly upregulated genes in the translatome were HSF1-dependent Hspa1a and Hspa1b , which encode the inducible HSP70. We found that HSP70 is upregulated in astrocyte endfeet after ischemia, coinciding with an increase in ubiquitination across the proteome. These findings suggest a robust proteostasis response to proteotoxic stress in the endfoot translatome after ischemia. Modulating proteostasis in endfeet may be a strategy to preserve endfeet function and BBB integrity after ischemic stroke.

In human stroke, brain swelling is an important predictor of neurological outcome and mortality, yet treatments to reduce or prevent brain swelling are extremely limited, due in part to an inadequate understanding of mechanisms. In preclinical studies on cerebroprotection in animal models of stroke, historically, the focus has been on reducing infarct size, and in most studies, a reduction in infarct size has been associated with a corresponding reduction in brain swelling. Unfortunately, such findings on brain swelling have little translational value for treating brain swelling in patients with stroke. This is because, in humans, brain swelling usually becomes evident, either symptomatically or radiologically, days after the infarct size has stabilized, requiring that the prevention or treatment of brain swelling target mechanism(s) that are independent of a reduction in infarct size. In this problematizing review, we highlight the often-neglected concept that brain edema and brain swelling are not simply secondary, correlative phenomena of stroke but distinct pathological entities with unique molecular and cellular mechanisms that are worthy of direct targeting. We outline the advances in approaches for the study of brain swelling that are independent of a reduction in infarct size. Although straightforward, the approaches reviewed in this study have important translational relevance for identifying novel treatment targets for post-ischemic brain swelling.

Brain swelling is a major cause of death and disability in ischemic stroke. Drugs of the gliflozin class, which target the Na + -coupled D-glucose cotransporter, SGLT2, are approved for type 2 diabetes mellitus (T2DM) and may be beneficial in other conditions, but data in cerebral ischemia are limited. We studied murine models of cerebral ischemia with middle cerebral artery occlusion/reperfusion (MCAo/R). Slc5a2 /SGLT2 mRNA and protein were upregulated de novo in astrocytes. Live cell imaging of brain slices from mice following MCAo/R showed that astrocytes responded to modest increases in D-glucose by increasing intracellular Na + and cell volume (cytotoxic edema), both of which were inhibited by the SGLT2 inhibitor, canagliflozin. The effect of canagliflozin was studied in three mouse models of stroke: non-diabetic and T2DM mice with a moderate ischemic insult (MCAo/R, 1/24 h) and non-diabetic mice with a severe ischemic insult (MCAo/R, 2/24 h). Canagliflozin reduced infarct volumes in models with moderate but not severe ischemic insults. However, canagliflozin significantly reduced hemispheric swelling and improved neurological function in all models tested. The ability of canagliflozin to reduce brain swelling regardless of an effect on infarct size has important translational implications, especially in large ischemic strokes.

Over 38 million people worldwide are living with HIV/AIDS, and more than half of them are affected by HIV-associated neurocognitive disorders (HAND). Such disorders are characterized by chronic neuroinflammation, neurotoxicity, and central nervous system deterioration, which lead to short- or long-term memory loss, cognitive impairment, and motor skill deficits that may show gender disparities. However, the underlying mechanisms remain unclear. Our previous study suggested that HIV-1 infection and viral protein R (Vpr) upregulate the SUR1-TRPM4 channel associated with neuroinflammation, which may contribute to HAND. The present study aimed to explore this relationship in a mouse model of HAND. This study employed the HIV transgenic Tg26 mouse model, comparing Tg26 mice with wildtype mice in various cognitive behavioral and memory tests, including locomotor activity tests, recognition memory tests, and spatial learning and memory tests. The study found that Tg26 mice exhibited impaired cognitive skills and reduced learning abilities compared to wildtype mice, particularly in spatial memory. Interestingly, male Tg26 mice displayed significant differences in spatial memory losses ( p < 0.001), while no significant differences were identified in female mice. Consistent with our early results, SUR1-TRPM4 channels were upregulated in Tg26 mice along with glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4), consistent with reactive astrocytosis and neuroinflammation. Corresponding reductions in neurosynaptic responses, as indicated by downregulation of Synapsin-1 (SYN1) and Synaptophysin (SYP), suggested synaptopathy as a possible mechanism underlying cognitive and motor skill deficits. In conclusion, our study suggests a possible relationship between SUR1-TRPM4-mediated neuroinflammation and synaptopathy with impairments of learning and memory in mice with HAND. These findings could help to develop new therapeutic strategies for individuals living with HAND.

Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na + /Ca 2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na + through SUR1-TRPM4 induced Ca 2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca 2+ . This increase in Ca 2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress., (© 2023. The Author(s).)

Spinal cord injury (SCI) is a devastating neurological condition with no effective treatment. Hypothermia induced by physical means (cold fluid) is established as an effective therapy in animal models of SCI, but its clinical translation to humans is hampered by several constraints. Hypothermia induced pharmacologically may be noninferior or superior to physically induced hypothermia for rapid, convenient systemic temperature reduction, but it has not been investigated previously in animal models of SCI. We used a rat model of SCI to compare outcomes in three groups: (1) normothermic controls; (2) hypothermia induced by conventional physical means; (3) hypothermia induced by intravenous (IV) dihydrocapsaicin (DHC). Male rats underwent unilateral lower cervical SCI and were treated after a 4-hour delay with physical cooling or IV DHC (∼0.60 mg/kg total) cooling (both 33.0 ± 1.0°C) lasting 4 hours; controls were kept normothermic. Telemetry was used to monitor temperature and heart rate during and after treatments. In two separate experiments, one ending at 48 hours, the other at 6 weeks, "blinded" investigators evaluated rats in the three groups for neurological function followed by histopathological evaluation of spinal cord tissues. DHC reliably induced systemic cooling to 32-33°C. At both the time points examined, the two modes of hypothermia yielded similar improvements in neurological function and lesion size compared with normothermic controls. Our results indicate that DHC-induced hypothermia may be comparable with physical hypothermia in efficacy, but more clinically feasible to administer than physical hypothermia.

Background: Gulf War (GW) Illness (GWI) is a debilitating condition with a complex constellation of immune, endocrine and neurological symptoms, including cognitive impairment, anxiety and depression. We studied a novel model of GWI based on 3 known common GW exposures (GWE): (i) intranasal lipopolysaccharide, to which personnel were exposed during desert sand storms; (ii) pyridostigmine bromide, used as prophylaxis against chemical warfare; and (iii) chronic unpredictable stress, an inescapable element of war. We used this model to evaluate prophylactic treatment with the PPARγ agonist, rosiglitazone (ROSI)., Methods: Rats were subjected to the three GWE for 33 days. In series 1 and 2, male and female GWE-rats were compared to naïve rats. In series 3, male rats with GWE were randomly assigned to prophylactic treatment with ROSI (GWE-ROSI) or vehicle. After the 33-day exposures, three neurofunctional domains were evaluated: cognition (novel object recognition), anxiety-like behaviors (elevated plus maze, open field) and depression-like behaviors (coat state, sucrose preference, splash test, tail suspension and forced swim). Brains were analyzed for astrocytic and microglial activation and neuroinflammation (GFAP, Iba1, tumor necrosis factor and translocator protein). Neurofunctional data from rats with similar exposures were pooled into 3 groups: naïve, GWE and GWE-ROSI., Results: Compared to naïve rats, GWE-rats showed significant abnormalities in the three neurofunctional domains, along with significant neuroinflammation in amygdala and hippocampus. There were no differences between males and females with GWE. GWE-ROSI rats showed significant attenuation of neuroinflammation and of some of the neurofunctional abnormalities., Conclusion: This novel GWI model recapitulates critical neurofunctional abnormalities reported by Veterans with GWI. Concurrent prophylactic treatment with ROSI was beneficial in this model., Competing Interests: The authors have declared that no competing interests exist.

Explosive blast-induced traumatic brain injury (blast-TBI) in military personnel is a leading cause of injury and persistent neurological abnormalities, including chronic pain. We previously demonstrated that chronic pain after spinal cord injury results from central sensitization in the posterior thalamus (PO). The presence of persistent headaches and back pain in veterans with blast-TBI suggests a similar involvement of thalamic sensitization. Here, we tested the hypothesis that pain after blast-TBI is associated with abnormal increases in activity of neurons in PO thalamus. We developed a novel model with two unique features: (1) blast-TBI was performed in awake, un-anesthetized rats, to simulate the human experience and to eliminate confounds of anesthesia and surgery inherent in other models; (2) only the cranium, rather than the entire body, was exposed to a collimated blast wave, with the blast wave striking the posterior cranium in the region of the occipital crest and foramen magnum. Three weeks after blast-TBI, rats developed persistent, ongoing spontaneous pain. Contrary to our hypothesis, we found no significant differences in the activity of PO neurons, or of neurons in the spinal trigeminal nucleus. There were also no significant changes in gliosis in either of these structures. This novel model will allow future studies on the pathophysiology of chronic pain after blast-TBI.

Background: Spontaneous intracerebral hemorrhage (ICH) leaves most survivors dependent at follow-up. The importance of promoting M2-like microglial responses is increasingly recognized as a key element to ameliorate brain injury following ICH. The osmotherapeutic agents, mannitol and hypertonic saline (HTS), which are routinely used to reduce intracranial pressure, have been shown to reduce neuroinflammation in experimental ischemic and traumatic brain injury, but anti-inflammatory effects of osmotherapies have not been investigated in ICH., Methods: We studied the effects of iso-osmotic mannitol and HTS in rat models of ICH utilizing high-dose and moderate-dose collagenase injections into the basal ganglia, associated with high and low mortality, respectively. We studied the effects of osmotherapies, first given 5 h after ICH induction, and then administered every 12 h thereafter (4 doses total). Immunohistochemistry was used to quantify microglial activation and polarization., Results: Compared to controls, mannitol and HTS increased plasma osmolarity 1 h after infusion (301 ± 1.5, 315 ± 4.2 and 310 ± 2.0 mOsm/kg, respectively), reduced mortality at 48 h (82, 36 and 53%, respectively), and reduced hemispheric swelling at 48 h (32, 21, and 17%, respectively). In both perihematomal and contralateral tissues, mannitol and HTS reduced activation of microglia/macrophages (abundance and morphology of Iba1 + cells), and in perihematomal tissues, they reduced markers of the microglia/macrophage M1-like phenotype (nuclear p65, TNF, and NOS2), increased markers of the microglia/macrophage M2-like phenotype (arginase, YM1, and pSTAT3), and reduced infiltration of CD45 + cells., Conclusions: Repeated dosing of osmotherapeutics at regular intervals may be a useful adjunct to reduce neuroinflammation following ICH.

Primary blast traumatic brain injury (bTBI) accounts for a significant proportion of wartime trauma. Previous studies have demonstrated direct brain injury by blast waves, but the effect of the location of the blast epicenter on the skull with regard to brain injury remains poorly characterized. In order to investigate the role of the blast epicenter location, we modified a previously established rodent model of cranium-only bTBI to evaluate two specific blast foci: a rostrally focused blast centered on bregma (B-bTBI), which excluded the foramen magnum region, and a caudally focused blast centered on the occipital crest, which included the foramen magnum region (FM-bTBI). At all blast overpressures studied (668-1880 kPa), rats subjected to FM-bTBI demonstrated strikingly higher mortality, increased durations of both apnea and hypoxia, and increased severity of convexity subdural hematomas, than rats subjected to B-bTBI. Together, these data suggest a unique role for the foramen magnum region in mortality and brain injury following blast exposure, and emphasize the importance of the choice of blast focus location in experimental models of bTBI.

[This corrects the article DOI: 10.1371/journal.pone.0171163.].

Blast-induced traumatic brain injury (blast-TBI) is associated with vestibulomotor dysfunction, persistent post-traumatic headaches and post-traumatic stress disorder, requiring extensive treatments and reducing quality-of-life. Treatment and prevention of these devastating outcomes require an understanding of their underlying pathophysiology through studies that take advantage of animal models. Here, we report that cranium-directed blast-TBI in rats results in signs of pain that last at least 8 weeks after injury. These occur without significantly elevated behavioural markers of anxiety-like conditions and are not associated with glial up-regulation in sensory thalamic nuclei. These injuries also produce transient vestibulomotor abnormalities that resolve within 3 weeks of injury. Thus, blast-TBI in rats recapitulates aspects of the human condition.

Blast traumatic brain injury (bTBI) affects both military and civilian populations, and often results in chronic deficits in cognition and memory. Chronic glial activation after bTBI has been linked with cognitive decline. Pharmacological inhibition of sulfonylurea receptor 1 (SUR1) with glibenclamide was shown previously to reduce glial activation and improve cognition in contusive models of CNS trauma, but has not been examined in bTBI. We postulated that glibenclamide would reduce chronic glial activation and improve long-term memory function after bTBI. Using a rat direct cranial model of bTBI (dc-bTBI), we evaluated the efficacy of two glibenclamide treatment paradigms: glibenclamide prophylaxis (pre-treatment), and treatment with glibenclamide starting after dc-bTBI (post-treatment). Our results show that dc-bTBI caused hippocampal astrocyte and microglial/macrophage activation that was associated with hippocampal memory dysfunction (rapid place learning paradigm) at 28days, and that glibenclamide pre-treatment, but not post-treatment, effectively protected against glial activation and memory dysfunction. We also report that a brief transient time-window of blood-brain barrier (BBB) disruption occurs after dc-bTBI, and we speculate that glibenclamide, which is mostly protein bound and does not normally traverse the intact BBB, can undergo CNS delivery only during this brief transient opening of the BBB. Together, our findings indicate that prophylactic glibenclamide treatment may help to protect against chronic cognitive sequelae of bTBI in warfighters and other at-risk populations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: In adult humans, cerebral microbleeds play important roles in neurodegenerative diseases but in neonates, the consequences of cerebral microbleeds are unknown. In rats, a single pro-angiogenic stimulus in utero predisposes to cerebral microbleeds after birth at term, a time when late oligodendrocyte progenitors (pre-oligodendrocytes) dominate in the rat brain. We hypothesized that two independent pro-angiogenic stimuli in utero would be associated with a high likelihood of perinatal microbleeds that would be severely damaging to white matter., Methods: Pregnant Wistar rats were subjected to intrauterine ischemia (IUI) and low-dose maternal lipopolysaccharide (mLPS) at embryonic day (E) 19. Pups were born vaginally or abdominally at E21-22. Brains were evaluated for angiogenic markers, microhemorrhages, myelination and axonal development. Neurological function was assessed out to 6 weeks., Results: mRNA (Vegf, Cd31, Mmp2, Mmp9, Timp1, Timp2) and protein (CD31, MMP2, MMP9) for angiogenic markers, in situ proteolytic activity, and collagen IV immunoreactivity were altered, consistent with an angiogenic response. Vaginally delivered pups exposed to prenatal IUI+mLPS had spontaneous cerebral microbleeds, abnormal neurological function, and dysmorphic, hypomyelinated white matter and axonopathy. Pups exposed to the same pro-angiogenic stimuli in utero but delivered abdominally had minimal cerebral microbleeds, preserved myelination and axonal development, and neurological function similar to naïve controls., Conclusions: In rats, pro-angiogenic stimuli in utero can predispose to vascular fragility and lead to cerebral microbleeds. The study of microbleeds in the neonatal rat brain at full gestation may give insights into the consequences of microbleeds in human preterm infants during critical periods of white matter development., Competing Interests: The authors have declared that no competing interests exist.

Recent preclinical studies have identified three treatments that are especially promising for reducing acute lesion expansion following traumatic spinal cord injury (SCI): riluzole, systemic hypothermia, and glibenclamide. Each has demonstrated efficacy in multiple studies with independent replication, but there is no way to compare them in terms of efficacy or safety, since different models were used, different laboratories were involved, and different outcomes were evaluated. Here, using a model of lower cervical hemicord contusion, we compared safety and efficacy for the three treatments, administered beginning 4 h after trauma. Treatment-associated mortality was 30% (3/10), 30% (3/10), 12.5% (1/8), and 0% (0/7) in the control, riluzole, hypothermia, and glibenclamide groups, respectively. For survivors, all three treatments showed overall favorable efficacy, compared with controls. On open-field locomotor scores (modified Basso, Beattie, and Bresnahan scores), hypothermia- and glibenclamide-treated animals were largely indistinguishable throughout the study, whereas riluzole-treated rats underperformed for the first two weeks; during the last four weeks, scores for the three treatments were similar, and significantly different from controls. On beam balance, hypothermia and glibenclamide treatments showed significant advantages over riluzole. After trauma, rats in the glibenclamide group rapidly regained a normal pattern of weight gain that differed markedly and significantly from that in all other groups. Lesion volumes at six weeks were: 4.8±0.7, 3.5±0.4, 3.1±0.3 and 2.5±0.3 mm(3) in the control, riluzole, hypothermia, and glibenclamide groups, respectively; measurements of spared spinal cord tissue confirmed these results. Overall, in terms of safety and efficacy, systemic hypothermia and glibenclamide were superior to riluzole.

Explosive blast-related injuries are one of the hallmark injuries of veterans returning from recent wars, but the effects of a blast overpressure on the brain are poorly understood. In this study, we used in vivo diffusion kurtosis imaging (DKI) and proton magnetic resonance spectroscopy (MRS) to investigate tissue microstructure and metabolic changes in a novel, direct cranial blast traumatic brain injury (dc-bTBI) rat model. Imaging was performed on rats before injury and 1, 7, 14 and 28 days after blast exposure (~517 kPa peak overpressure to the dorsum of the head). No brain parenchyma abnormalities were visible on conventional T2-weighted MRI, but microstructural and metabolic changes were observed with DKI and proton MRS, respectively. Increased mean kurtosis, which peaked at 21 days post injury, was observed in the hippocampus and the internal capsule. Concomitant increases in myo-Inositol (Ins) and Taurine (Tau) were also observed in the hippocampus, while early changes at 1 day in the Glutamine (Gln) were observed in the internal capsule, all indicating glial abnormality in these regions. Neurofunctional testing on a separate but similarly treated group of rats showed early disturbances in vestibulomotor functions (days 1-14), which were associated with imaging changes in the internal capsule. Delayed impairments in spatial memory and in rapid learning, as assessed by Morris Water Maze paradigms (days 14-19), were associated with delayed changes in the hippocampus. Significant microglial activation and neurodegeneration were observed at 28 days in the hippocampus. Overall, our findings indicate delayed neurofunctional and pathological abnormalities following dc-bTBI that are silent on conventional T2-weighted imaging, but are detectable using DKI and proton MRS.

Traumatic brain injury (TBI) caused by an explosive blast (blast-TBI) is postulated to result, in part, from transvascular transmission to the brain of a hydrodynamic pulse (a.k.a., volumetric blood surge, ballistic pressure wave, hydrostatic shock, or hydraulic shock) induced in major intrathoracic blood vessels. This mechanism of blast-TBI has not been demonstrated directly. We tested the hypothesis that a blast wave impacting the thorax would induce a hydrodynamic pulse that would cause pathological changes in the brain. We constructed a Thorax-Only Blast Injury Apparatus (TOBIA) and a Jugular-Only Blast Injury Apparatus (JOBIA). TOBIA delivered a collimated blast wave to the right lateral thorax of a rat, precluding direct impact on the cranium. JOBIA delivered a blast wave to the fluid-filled port of an extracorporeal intravenous infusion device whose catheter was inserted retrograde into the jugular vein, precluding lung injury. Long Evans rats were subjected to sublethal injury by TOBIA or JOBIA. Blast injury induced by TOBIA was characterized by apnea and diffuse bilateral hemorrhagic injury to the lungs associated with a transient reduction in pulse oximetry signals. Immunolabeling 24 h after injury by TOBIA showed up-regulation of tumor necrosis factor alpha, ED-1, sulfonylurea receptor 1 (Sur1), and glial fibrillary acidic protein in veins or perivenular tissues and microvessels throughout the brain. The perivenular inflammatory effects induced by TOBIA were prevented by ligating the jugular vein and were reproduced using JOBIA. We conclude that blast injury to the thorax leads to perivenular inflammation, Sur1 up-regulation, and reactive astrocytosis resulting from the induction of a hydrodynamic pulse in the vasculature.

Both glibenclamide and riluzole reduce necrosis and improve outcome in rat models of spinal cord injury (SCI). In SCI, gene suppression experiments show that newly upregulated sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channels in microvascular endothelial cells are responsible for "persistent sodium currents" that cause capillary fragmentation and "progressive hemorrhagic necrosis". Glibenclamide is a potent blocker of Sur1-regulated NC(Ca-ATP) channels (IC(50), 6-48 nM). Riluzole is a pleotropic drug that blocks "persistent sodium currents" in neurons, but in SCI, its molecular mechanism of action is uncertain. We hypothesized that riluzole might block the putative pore-forming subunits of Sur1-regulated NC(Ca-ATP) channels, Trpm4. In patch clamp experiments, riluzole blocked Sur1-regulated NC(Ca-ATP) channels in endothelial cells and heterologously expressed Trpm4 (IC(50), 31 μM). Using a rat model of cervical SCI associated with high mortality, we compared the effects of glibenclamide and riluzole administered beginning at 3h and continuing for 7 days after impact. During the acute phase, both drugs reduced capillary fragmentation and progressive hemorrhagic necrosis, and both prevented death. At 6 weeks, modified (unilateral) Basso, Beattie, Bresnahan locomotor scores were similar, but measures of complex function (grip strength, rearing, accelerating rotarod) and tissue sparing were significantly better with glibenclamide than with riluzole. We conclude that both drugs act similarly, glibenclamide on the regulatory subunit, and riluzole on the putative pore-forming subunit of the Sur1-regulated NC(Ca-ATP) channel. Differences in specificity, dose-limiting potency, or in spectrum of action may account for the apparent superiority of glibenclamide over riluzole in this model of severe SCI., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Traumatic brain injury resulting from an explosive blast is one of the most serious wounds suffered by warfighters, yet the effects of explosive blast overpressure directly impacting the head are poorly understood. We developed a rodent model of direct cranial blast injury (dcBI), in which a blast overpressure could be delivered exclusively to the head, precluding indirect brain injury via thoracic transmission of the blast wave. We constructed and validated a Cranium Only Blast Injury Apparatus (COBIA) to deliver blast overpressures generated by detonating .22 caliber cartridges of smokeless powder. Blast waveforms generated by COBIA replicated those recorded within armored vehicles penetrated by munitions. Lethal dcBI (LD(50) ∼ 515 kPa) was associated with: (1) apparent brainstem failure, characterized by immediate opisthotonus and apnea leading to cardiac arrest that could not be overcome by cardiopulmonary resuscitation; (2) widespread subarachnoid hemorrhages without cortical contusions or intracerebral or intraventricular hemorrhages; and (3) no pulmonary abnormalities. Sub-lethal dcBI was associated with: (1) apnea lasting up to 15 sec, with transient abnormalities in oxygen saturation; (2) very few delayed deaths; (3) subarachnoid hemorrhages, especially in the path of the blast wave; (4) abnormal immunolabeling for IgG, cleaved caspase-3, and β-amyloid precursor protein (β-APP), and staining for Fluoro-Jade C, all in deep brain regions away from the subarachnoid hemorrhages, but in the path of the blast wave; and (5) abnormalities on the accelerating Rotarod that persisted for the 1 week period of observation. We conclude that exposure of the head alone to severe explosive blast predisposes to significant neurological dysfunction.

Background: There is little that can be done to treat or reverse the primary injury that occurs at the time of a traumatic brain injury (TBI). Initial management of the patient with severe TBI focuses on prevention of subsequent secondary insults, namely, intracranial hypertension (ICH) and cerebral hypoperfusion (CH). Currently, there is no reliable way to predict which patients will develop ICH and CH other than clinical acumen; therefore, indicators of impending secondary intracranial insults may be useful in predicting these events and allowing for prevention and early intervention. This study was undertaken to investigate the relationship of cytokine levels with intracranial pressure (ICP) and cerebral perfusion pressure (CPP) in patients with severe TBI., Methods: Patients at the R Adams Cowley Shock Trauma Center were prospectively enrolled for a 6-month period. Inclusion criteria were older than 17 years, admission within the first 6 hours after injury, Glasgow Coma Scale<9 on admission, and placement of a clinically indicated ICP monitor. Serum and cerebrospinal fluid, when available, were collected on admission and twice daily for 7 days. Cytokine levels of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α were analyzed by multiplex bead array assays. Hourly values for ICP and CPP were recorded, and means, minimum (for CPP) or maximum (for ICP) values, percentage time ICP>20 mm Hg (%ICP20) and CPP<60 mm Hg (%CPP60), and cumulative Pressure Times Time Dose (PTD; mm Hg·h) for ICP>20 mm Hg (PTD ICP20) and CPP<60 mm Hg (PTD CPP60) were compared with the serum and cerebrospinal fluid levels that were drawn before 12-hour time periods (PRE) and after 12-hour time periods (POST) of monitoring., Results: Twenty-four patients were enrolled. In-hospital mortality was 12.5%, and good functional outcome was noted in 58%. Two hundred and seventy-five serum samples were taken and analyzed. IL-6 levels in the serum were found in the highest concentration of the cytokines measured. PTD ICP20 and PTD CPP60 were moderately correlated with increased PRE IL-8 levels (r=0.34, p<0.001; r=0.53, p<0.001). PTD ICP20 was also correlated with PRE TNF-α levels (r=0.27, p<0.001) as was PTD CPP60 (r=0.25, p<0.001). POST IL-8 levels were found to be correlated with PTD ICP20 (r=0.46, p<0.001) and PTD CPP60 (r=0.54, p<0.001). POST TNF-α was associated with PTD ICP20 (r=0.45, p<0.001). PTD CPP60 was also moderately correlated with POST TNF-α levels (r=0.26, p<0.001). When comparing patients with good versus poor outcome, median daily serum IL-8 levels were associated with poor outcome., Conclusions: IL-8 and, to a lesser extent, TNF-α demonstrated the most promise in this study to be candidate serum markers of impending ICH and CH. The clinical relevance of this is the suggestion that we may be able to predict impending secondary insults after TBI before the clinical manifestation of these events. Given the known morbidity of ICH and CH, early intervention and prevention may have a significant impact on outcome. This becomes even more important when decisions must be made about timing of interventions. Increased levels of IL-8 and TNF-α in the serum during episodes of ICH and CH imply there are significant systemic effects of these events. These serum biomarkers are promising as diagnostic targets. In addition, further study of the precise role of these molecules may have significant implications for inflammatory system manipulation in the management of severe TBI.

Our objective was to evaluate the hemostatic efficacy of a newly modified chitosan in a porcine grade V liver injury model. Fifteen Yorkshire pigs underwent standardized grade V liver injuries with a specially designed liver clamp and were randomized to either modified chitosan (MC) patch treatment or standard gauze packing. Free bleeding was allowed for 30 seconds. Fluid resuscitation was infused as necessary to reestablish a mean arterial pressure (MAP) within at least 80 per cent of the preinjury MAP. Animals were observed for 90 minutes or until death. Endpoints were survival, total blood loss, time to hemostasis, and resuscitation MAP, and resuscitation volume. Total mean blood loss was less in the MC patch group (464 +/- 267 mL vs 1234 +/- 78 mL, P < 0.001). Time to hemostasis was significantly less (4.8 +/- 2.5 minutes in the MC patch group vs 9.6 +/- 2.5 minutes, P < 0.01). Fluid resuscitation was less (1098 +/- 459 mL in the MC patch group vs 1770 +/- 172 mL, P < 0.01). Survival was 100 per cent in the MC patch group and 80 per cent in the gauze packing group. MC patches demonstrate the continued hemostatic agent evolution for improved control of lethal solid organ bleeding.

An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression ("blossoming") of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NC(Ca-ATP) channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI.

Frontal impact, closed head trauma is a frequent cause of traumatic brain injury (TBI) in motor vehicle and sports accidents. Diffuse axonal injury (DAI) is common in humans and experimental animals, and results from shearing forces that develop within the anisotropic brain. Because the specific anisotropic properties of the brain are axis-dependent, the anatomical site where force is applied as well as the resultant acceleration, be it linear, rotational, or some combination, are important determinants of the resulting pattern of brain injury. Available rodent models of closed head injury do not reproduce the frontal impact commonly encountered in humans. Here we describe a new rat model of closed head injury that is a modification of the impact-acceleration model of Marmarou. In our model (the Maryland model), the impact force is applied to the anterior part of the cranium and produces TBI by causing anterior-posterior plus sagittal rotational acceleration of the brain inside the intact cranium. Skull fractures, prolonged apnea, and mortality were absent. The animals exhibited petechial hemorrhages, DAI marked by a bead-like pattern of beta-amyloid precursor protein (beta-APP) in damaged axons, and widespread upregulation of beta-APP in neurons, with regions affected including the orbitofrontal cortex (coup), corpus callosum, caudate, putamen, thalamus, cerebellum, and brainstem. Activated caspase-3 was prominent in hippocampal neurons and Purkinje cells at the grey-white matter junction of the cerebellum. Neurobehavioral dysfunction, manifesting as reduced spontaneous exploration, lasted more than 1 week. We conclude that the Maryland model produces diffuse injuries that may be relevant to human brain injury.

Study Objective: Rapid hemostasis is crucial in controlling severe extremity hemorrhage. Our objective is to evaluate the hemostatic efficacy of a newly modified amylopectin powder in a model of severe extremity arterial hemorrhage., Methods: Anesthetized pigs underwent severe, reproducible femoral artery injuries. Animals were randomized (nonblinded) to either modified amylopectin powder (n=10) or standard gauze application (n=6). Each hemostatic agent was applied through a pool of blood with manual compression for 3-minute intervals until hemostasis was achieved. Fluid resuscitation was infused as necessary to reestablish a mean arterial pressure within at least 80% of the preinjury mean arterial pressure if possible. The primary measured outcome was total blood loss. Secondary endpoints were survival, time to hemostasis, resuscitation mean arterial pressure, and resuscitation volume., Results: Pretreatment blood losses were similar in both groups. Median (absolute average deviation of the median) posttreatment blood loss was significantly less in the modified amylopectin powder group than in the gauze group, 275 (108) mL versus 1,312 (171) mL. Resuscitation mean arterial pressure at 180 minutes after injury was 68% of preinjury mean arterial pressure in the modified amylopectin powder group and undetectable in all control animals. Fluid volume required for resuscitation was 1,962 (258) mL in the modified amylopectin powder group and 2,875 (150) mL in the gauze group. Time to hemostasis was 9.0 (2.1) minutes in the modified amylopectin powder group. Hemostasis was not achieved in any animal in the gauze group. Survival was 100% in the modified amylopectin powder group, whereas no animals survived in the gauze group., Conclusion: Modified amylopectin powder demonstrates the ability to control major vascular bleeding in a lethal arterial injury model during a 3-hour period.

Background: Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found in the cytoskeleton of astroglia. Recent work has indicated that GFAP may serve as a serum marker of traumatic brain injury (TBI) that is released after central nervous system cell damage., Methods: Serum from 51 critically injured trauma patients was prospectively collected on admission and on hospital day 2. All patients underwent an admission head computed tomography (CT) scan as a part of their clinical evaluation. Patients with facial fractures in the absence of documented TBI and patients with spinal cord injury were excluded. Demographic and outcome data were collected prospectively. Serum GFAP was measured in duplicate using enzyme-linked immunosorbent assay techniques., Results: Thirty-nine (76%) of the 51 patients had CT-documented TBI. The study cohort was 72.5% men with a mean age of 43 years and mean Injury Severity Score (ISS) of 30.2. There were no statistically significant demographic differences between the two groups. At admission day, the mean GFAP level in non-TBI patients was 0.07 pg/mL compared with 6.77 pg/mL in TBI patients (p = 0.002). On day 2 the mean GFAP level was 0.02 in non-TBI patients compared with 2.17 in TBI patients (p = 0.003). Using regression analysis to control for age, sex, and ISS, the Head Abbreviated Injury Scale was predictive of the level of GFAP on both days 1 and 2 (p values 0.006 and 0.026, respectively). Although GFAP levels were not predictive of increased hospital length of stay, intensive care unit length of stay, or ventilator days, high GFAP levels on hospital day 2 were predictive of mortality when controlling for age, sex, and ISS (odds ratio 1.45, p value 0.028). The area under the receiver operating characteristic curve for GFAP was 0.90 for day 1 and 0.88 for day 2. A GFAP cutoff point of 1 pg/mL yielded 100% specificity and 50% to 60% sensitivity for TBI., Conclusions: GFAP is a serum marker of TBI, and persistent elevation on day 2 is predictive of increased mortality. Excellent specificity for CT-documented brain injury was found using a cutoff point of 1 pg/mL.

Background: In low concentrations, lipopolysaccharide-binding protein (LBP), an acute-phase protein recognizing lipopolysaccharide (LPS), catalyzes its transfer to the cellular receptor consisting of CD14 and Toll-like receptor-4. Previous studies have documented increased serum LBP concentrations in patients with sepsis, systemic inflammatory response syndrome (SIRS), or acute pancreatitis and after cardiopulmonary bypass. No prior studies have examined LBP expression in trauma victims. We hypothesized that admission LBP plasma concentrations are predictive of outcome (mortality) in trauma. This study assessed time-dependent changes in serum LBP concentrations in trauma patients soon after injury., Methods: A prospective, single-institution, observational cohort study of 121 adult trauma patients (age > or =17 years) with moderate to severe injury who required hospitalization. The trauma patients were male in 79.6% of the cases and had a mean age of 43.0 +/- 20.6 years. The mean injury severity score (ISS) was 23 +/- 12, and the crystalloid resuscitation volume given in the first 24 h averaged 6,640 +/- 3,729 mL. Informed consent was obtained on admission, and blood samples were drawn on admission and at 24 h postadmission. Prospective data were collected for daily SIRS score, multiple organ dysfunction score (MODS), and sequential organ failure assessment (SOFA) score, complications, and outcomes. Plasma concentrations of LBP were measured by enzyme-linked immunosorbent assay., Results: Sixty patients (48.8% of the study cohort) required emergency surgical intervention and sustained a substantial intraoperative blood loss (mean 1,404 +/- 2,757 mL). The hospital mortality rate was 16.3% (20 patients). The mean intensive care unit stay was 8.9 +/- 16.4 days, and the hospital stay was 14.8 +/- 19.6 days. The patients had a significantly higher serum concentrations of LBP on admission (mean 28.0 +/- 25.3 mg/L; range 2-100 mg/L) than did control subjects (mean 6.2 +/- 2.1 mg/L; range 1.3-12.8 mg/L; p < 0.01), similar to the plasma concentrations previously reported in septic patients. A significant increase in LBP concentration was noted at 24 h (mean 72.3 +/- 45.7 mg/L; range 8-210 mg/L; p < 0.05). The admission LBP concentration was significantly greater in nonsurvivors than in survivors. However, after controlling for age and ISS, the admission LBP concentration did not predict death.

The quinazoline-derived alpha1-adrenoceptor antagonists, doxazosin and terazosin have been recently shown to induce an anoikis effect in human prostate cancer cells and to suppress prostate tumor vascularity in clinical specimens [Keledjian and Kyprianou, 2003]. This study sought to examine the ability of doxazosin to affect the growth of human vascular endothelial cells and to modulate vascular endothelial growth factor (VEGF)-mediated angiogenesis. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro model to determine the effect of doxazosin on cell growth, apoptosis, adhesion, migration, and angiogenic response of endothelial cells. The effect of doxazosin on cell viability and apoptosis induction of human endothelial cells, was evaluated on the basis of trypan blue and Hoechst 33342 staining, respectively. Doxazosin antagonized the VEGF-mediated angiogenic response of HUVEC cells, by abrogating cell adhesion to fibronectin and collagen-coated surfaces and inhibiting cell migration, via a potential downregulation of VEGF expression. Furthermore there was a significant suppression of in vitro angiogenesis by doxazosin on the basis of VEGF-mediated endothelial tube formation (P < 0.01). Fibroblast growth factor-2 (FGF-2) significantly enhanced HUVEC cell tube formation (P < 0.01) and this effect was suppressed by doxazosin. These findings provide new insight into the ability of doxazosin to suppress the growth and angiogenic response of human endothelial cells by interfering with VEGF and FGF-2 action. This evidence may have potential therapeutic significance in using this quinazoline-based compound as an antiangiogenic agent for the treatment of advanced prostate cancer., (2004 Wiley-Liss, Inc.)

Purpose: The ability of the quinazoline derived alpha1-adrenoceptor antagonists doxazosin and terazosin to induce apoptosis in benign and malignant prostate cells has been established. In this study we investigated the effect of the 2 piperazidinyl quinazoline based alpha1-adrenoceptor antagonists and the methoxybenzene sulfonamide alpha1-antagonist tamsulosin on human prostate cancer cell adhesion., Materials and Methods: Androgen independent PC-3 prostate cancer cells and PC-3 transfectant clones over expressing the apoptosis suppressor bcl-2 were used as an in vitro model. Cells were treated with pharmacologically relevant doses of 1 of the 3 alpha1-adrenoceptor antagonists and the effect on cell viability/cell adhesion on various substrates was examined. Analysis of expression of key attachment factors, such as vascular endothelial growth factor (VEGF) and hypoxia inducible factor-alpha, was performed., Results: Our results indicate a significant decrease in prostate cancer cell adhesion to gelatin and collagen but not to fibronectin in prostate cancer cells treated with doxazosin or terazosin (25 microM.) compared with untreated control cultures (p <0.05). In contrast, tamsulosin had no effect on prostate cancer cell adhesion. The 2 quinazolines doxazosin and terazosin but not tamsulosin had a significant inhibitory effect on prostate tumor cell invasion. In bcl-2 over expressing prostate cancer cells there was significant suppression of doxazosin induced anoikis and cell invasion compared with neocontrol transfectants (p <0.05). Doxazosin resulted in transient down-regulation (2-fold decrease) of VEGF at the mRNA and protein levels, as detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. No significant changes in the expression profile of hypoxia inducible factor-1 alpha were observed after treatment with quinazolines. Furthermore, bcl-2 resulted in partial reversion of the doxazosin induced VEGF decrease., Conclusions: These findings demonstrate that the quinazoline derived alpha1-antagonists doxazosin and terazosin but not sulfonamide based tamsulosin induce anoikis and inhibit prostate cancer cell invasion, an effect that is antagonized by bcl-2. This molecular basis of an alpha1-adrenoceptor independent action against prostate cancer cells by the quinazolines may have potential therapeutic significance in prostate cancer.

Background: We previously demonstrated that the quinazoline-derived a1-adrenoceptor antagonists doxazosin and terazosin suppress prostate cancer growth via apoptosis induction. The aim of this study was to determine the potential effect of a1-adrenoceptor antagonists on tumor vascularity of the human prostate., Methods: A total of 34 men with benign prostatic hyperplasia (BPH) who have been on terazosin treatment (for the obstructive symptoms) were pathologically diagnosed with prostate cancer following surgery. These patients were stratified according to the length of treatment periods with terazosin into two groups, 1 week-6 months, and 6-17 months. The control group consisted of prostatectomy specimens from 25 untreated prostate cancer patients undergoing surgery for localized disease. Formalin-fixed, paraffin-embedded prostate specimens were analyzed for apoptosis (TUNEL assay), cell proliferation (Ki-67), microvessel density (MVD) (von Willebrand factor/Factor VIII), vascular endothelial growth factor (VEGF) expression, and prostate specific antigen (PSA) immunoreactivity., Results: A significant induction of apoptosis was observed among cancerous prostatic epithelial cells in the terazosin-treated, as compared to the untreated prostate cancer specimens, while there was no significant change in the proliferative index of the same tumor cell populations after treatment. Furthermore, terazosin resulted in a significant decrease in prostate tissue MVD compared with the untreated group (P < 0.01), that correlated with the increased apoptotic index of the cancerous areas. Tissue PSA expression in the prostatic tumor foci was also markedly reduced after terazosin treatment, while no significant changes in VEGF expression were detected., Conclusions: These findings provide the first evidence that terazosin, a quinazoline-based a1-blocker decreases prostate tumor vascularity. Our study has significant clinical implications in identifying selected alpha1-adrenoceptor antagonists as potential anti-tumor agents with apoptotic and anti-angiogenic effects in the human prostate that can be exploited for the treatment of advanced prostate cancer.