tacrolimus and Brain-Injuries

Topics: Alzheimer Disease; Blindness, Cortical; Brain Diseases; Brain Injuries; Cerebral Infarction; Creutzfeldt-Jakob Syndrome; Cyclosporine; Electrodiagnosis; Humans; Immunosuppressive Agents; Occipital Lobe; Seizures; Tacrolimus; Visual Cortex

Neuroimmunophilin ligands are a class of compounds that hold great promise for the treatment of nerve injuries and neurological disease. In contrast to neurotrophins (e.g., nerve growth factor), these compounds readily cross the blood-brain barrier, being orally effective in a variety of animal models of ischaemia, traumatic nerve injury and human neurodegenerative disorders. A further distinction is that neuroimmunophilin ligands act via unique receptors that are unrelated to the classical neurotrophic receptors (e.g., trk), making it unlikely that clinical trials will encounter the same difficulties found with the neurotrophins. Another advantage is that two neuroimmunophilin ligands (cyclosporin A and FK-506) have already been used in humans (as immunosuppressant drugs). Whereas both cyclosporin A and FK-506 demonstrate neuroprotective actions, only FK-506 and its derivatives have been clearly shown to exhibit significant neuroregenerative activity. Accordingly, the neuroprotective and neuroregenerative properties seem to arise via different mechanisms. Furthermore, the neuroregenerative property does not involve calcineurin inhibition (essential for immunosuppression). This is important since most of the limiting side effects produced by these drugs arise via calcineurin inhibition. A major breakthrough for the development of this class of compounds for the treatment of human neurological disorders was the ability to separate the neuroregenerative property of FK-506 from its immunosuppressant action via the development of non-immunosuppressant (non-calcineurin inhibiting) derivatives. Further studies revealed that different receptor subtypes, or FK-506-binding proteins (FKBPs), mediate immunosuppression and nerve regeneration (FKBP-12 and FKBP-52, respectively, the latter being a component of steroid receptor complexes). Thus, steroid receptor chaperone proteins represent novel targets for future drug development of novel classes of compounds for the treatment of a variety of human neurological disorders, including traumatic injury (e.g., peripheral nerve and spinal cord), chemical exposure (e.g., vinca alkaloids, Taxol) and neurodegenerative disease (e.g. , diabetic neuropathy and Parkinson's disease).

Topics: Animals; Brain Injuries; Carpal Tunnel Syndrome; Cyclosporine; Humans; Immunophilins; Immunosuppressive Agents; Ligands; Nerve Regeneration; Nervous System Diseases; Spinal Cord Injuries; Tacrolimus; Tacrolimus Binding Protein 1A

This study aimed to investigate the protective effect of tacrolimus (FK506), an immunosuppressive agent, on secondary brain damage in rats with experimental head trauma.. 40 Sprague-Dawley rats, aged 10-12 weeks and weighing 250-350 g, were used without gender selection. The subjects that were divided into five groups of 8 rats per group (sham control, negative control, positive control, vehicle control, and treatment) were sacrificed 1 month after head trauma was induced under appropriate conditions, their brains were then removed en bloc and evaluated histopathologically. Secondary brain injury was evaluated with the immunoreactive score (IRS) after Glial Fibrillary Acid Protein staining of gliosis that would occur in brain tissue.. The evaluation of the histopathological IRS values of all groups showed significant statistical differences between all groups. The pairwise group comparison revealed the highest increase in IRS value in the treatment group (p<0.05), with no statistical significance despite the increase in the negative control, positive control, and vehicle control groups. The sham group had the lowest rate of severe histopathological reaction score.. It was observed that the group treated with FK506 had a statistically significant increase in gliosis in the traumatic area compared to the other control groups. This shows that FK506 cannot prevent and even increase gliosis by a mechanism that has not yet been clarified. In conclusion, it is obvious that the FK506 immunosuppressive agent does not reduce post-traumatic brain injury; on the contrary, it increases gliosis.

Topics: Animals; Brain Injuries; Gliosis; Immunosuppressive Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

Repetitive brain injury, particularly that occurring with sporting-related injuries, has recently garnered increased attention in both the clinical and public settings. In the laboratory, we have demonstrated the adverse axonal and vascular consequences of repetitive brain injury and have demonstrated that moderate hypothermia and/or FK506 exerted protective effects after repetitive mild traumatic brain injury (mTBI) when administered within a specific time frame, suggesting a range of therapeutic modalities to prevent a dramatic exacerbation. In this communication, we revisit the utility of targeted therapeutic intervention to seek the minimal level of hypothermia needed to achieve protection while probing the role of oxygen radicals and their therapeutic targeting. Male Sprague-Dawley rats were subjected to repetitive mTBI by impact acceleration injury. Mild hypothermia (35 °C, group 2), superoxide dismutase (group 3), and Tempol (group 4) were employed as therapeutic interventions administered 1  h after the repetitive mTBI. To assess vascular function, cerebral vascular reactivity to acetylcholine was evaluated 3 and 4 h after the repetitive mTBI, whereas to detect the burden of axonal damage, amyloid precursor protein (APP) density in the medullospinal junction was measured. Whereas complete impairment of vascular reactivity was observed in group 1 (without intervention), significant preservation of vascular reactivity was found in the other groups. Similarly, whereas remarkable increase in the APP-positive axon was observed in group 1, there were no significant increases in the other groups. Collectively, these findings indicate that even mild hypothermia or the blunting free radical damage, even when performed in a delayed period, is protective in repetitive mTBI.

Topics: Animals; Brain; Brain Injuries; Disease Models, Animal; Free Radical Scavengers; Hypothermia, Induced; Immunohistochemistry; Male; Rats; Rats, Sprague-Dawley; Tacrolimus

Recent interest in mild traumatic brain injury (mTBI) has increased the recognition that repetitive mTBI occurring within the sports and military settings can exacerbate the adverse consequences of the initial injury. While multiple studies have recently reported the pathological, metabolic, and functional changes associated with repetitive mTBI, no consideration has been given to the development of therapeutic approaches to attenuate these abnormalities. In this study, we used the model of repetitive impact acceleration insult previously reported by our laboratory to cause no initial structural and functional changes, yet evoke dramatic change following second insult of the same intensity. Using this model, we employed established neuroprotective agents including FK506 and hypothermia that were administered 1 h after the second insult. Following either therapeutic intervention, changes of cerebral vascular reactivity to acetylcholine were assessed through a cranial window. Following the completion of the vascular studies, the animals were prepared to access the numbers of amyloid precursor protein (APP) positive axons, a marker of axonal damage. Following repetitive injury, cerebral vascular reactivity was dramatically preserved by either therapeutic intervention or the combination thereof compared to control group in which no intervention was employed. Similarly, APP density was significantly lower in the therapeutic intervention group compared in controls. Although the individual use of FK506 or hypothermia exerted significant protection, no additive benefit was found when both therapies were combined. In sum, the current study demonstrates that the exacerbated pathophysiological changes associated with repetitive mTBI can be therapeutically targeted.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Brain Injuries; Capillaries; Cerebrovascular Circulation; Combined Modality Therapy; Hypothermia, Induced; Immunohistochemistry; Immunosuppressive Agents; Male; Rats; Rats, Sprague-Dawley; Tacrolimus

Traumatic brain injury (TBI) causes both an acute loss of tissue and a progressive injury through reactive processes such as excitotoxicity and inflammation. These processes may worsen neural dysfunction by altering neuronal circuitry beyond the focally-damaged tissue. One means of circuit alteration may involve dendritic spines, micron-sized protuberances of dendritic membrane that support most of the excitatory synapses in the brain. This study used a modified Golgi-Cox technique to track changes in spine density on the proximal dendrites of principal cells in rat forebrain regions. Spine density was assessed at 1 h, 24 h, and 1 week after a lateral fluid percussion TBI of moderate severity. At 1 h after TBI, no changes in spine density were observed in any of the brain regions examined. By 24 h after TBI, however, spine density had decreased in ipsilateral neocortex in layer II and III and dorsal dentate gyrus (dDG). This apparent loss of spines was prevented by a single, post-injury administration of the calcineurin inhibitor FK506. These results, together with those of a companion study, indicate an FK506-sensitive mechanism of dendritic spine loss in the TBI model. Furthermore, by 1 week after TBI, spine density had increased substantially above control levels, bilaterally in CA1 and CA3 and ipsilaterally in dDG. The apparent overgrowth of spines in CA1 is of particular interest, as it may explain previous reports of abnormal and potentially epileptogenic activity in this brain region.

Topics: Animals; Brain; Brain Injuries; Dendritic Spines; Disease Models, Animal; Male; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Tacrolimus

Diffuse axonal injury is a major component of traumatic brain injury in children and correlates with long-term cognitive impairment. Traumatic brain injury in adult rodents has been linked to a decrease in compound action potential (CAP) in the corpus callosum, but information on trauma-associated diffuse axonal injury in immature rodents is limited. We investigated the effects of closed head injury on CAP in the corpus callosum of 17-day-old rats. The injury resulted in CAP deficits of both myelinated and unmyelinated fibers in the corpus callosum between 1 and 14 days postinjury (dpi). These deficits were accompanied by intra-axonal dephosphorylation of the 200-kDa neurofilament subunit (NF200) at 1 and 3 dpi, a decrease in total NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi. Although total phosphatase activity decreased at 1 dpi, calcineurin activity was unchanged. The calcineurin inhibitor, FK506, significantly attenuated the injury-induced NF200 dephosphorylation of NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi but did not affect the decrease in NF200 protein levels or impaired axonal transport. FK506 had no effect on CAP deficits at 3 dpi but exacerbated the deficit in only the myelinated fibers at 7 dpi. Thus, in contrast to adult animals, FK506 treatment did not improve axonal function in brain-injured immature animals, suggesting that calcineurin may not contribute to impaired axonal function.

Topics: Aging; Animals; Axons; Brain Injuries; Female; Immunosuppressive Agents; Male; Rats; Rats, Sprague-Dawley; Tacrolimus

This study evaluated the utility of combinational therapy, coupling delayed posttraumatic hypothermia with delayed FK506 administration, on altered cerebral vascular reactivity, axonal injury, and blood-brain barrier (BBB) disruption seen following traumatic brain injury (TBI). Animals were injured, subjected to various combinations of hypothermic/FK506 intervention, and equipped with cranial windows to assess pial vascular reactivity to acetylcholine. Animals were then processed with antibodies to the amyloid precursor protein and immunoglobulin G to assess axonal injury and BBB disruption, respectively. Animals were assigned to five groups: (1) sham injury plus delayed FK506, (2) TBI, (3) TBI plus delayed hypothermia, (4) TBI plus delayed FK506, and (5) TBI plus delayed hypothermia with FK506. Sham injury plus FK506 had no impact on vascular reactivity, axonal injury, or BBB disruption. Traumatic brain injury induced dramatic axonal injury and altered pial vascular reactivity, while triggering local BBB disruption. Delayed hypothermia or FK506 after TBI provided limited protection. However, TBI with combinational therapy achieved significantly enhanced vascular and axonal protection, with no BBB protection. This study shows the benefits of combinational therapy, using posttraumatic hypothermia with FK506 to attenuate important features of TBI. This suggests that hypothermia not only protects but also extends the therapeutic window for improved FK506 efficacy.

Topics: Animals; Arterioles; Axons; Blood Pressure; Blood-Brain Barrier; Brain Injuries; Capillaries; Carbon Dioxide; Hydrogen-Ion Concentration; Hypothermia, Induced; Immunohistochemistry; Immunophilins; Immunosuppressive Agents; Male; Microcirculation; Oxygen; Rats; Rats, Sprague-Dawley; Tacrolimus

Cortical spreading depression (CSD) is associated with mitochondrial depolarization, increasing intracellular Ca(2+), and the release of free fatty acids, which favor opening of the mitochondrial permeability transition pore (mPTP) and activation of calcineurin (CaN). Here, we test the hypothesis that cyclosporine A (CsA), which blocks both mPTP and CaN, ameliorates the persistent reduction of cerebral blood flow (CBF), impaired vascular reactivity, and a persistent rise in the cerebral metabolic rate of oxygen (CMRO(2)) following CSD. In addition to CsA, we used the specific mPTP blocker NIM811 and the specific CaN blocker FK506. Cortical spreading depression was induced in rat frontal cortex. Electrocortical activity was recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension with polarographic microelectrodes. Electrocortical activity, basal CBF, CMRO(2), and neurovascular and neurometabolic coupling were unaffected by all three drugs under control conditions. NIM811 augmented the rise in CBF observed during CSD. Cyclosporine A and FK506 ameliorated the persistent decrease in CBF after CSD. All three drugs prevented disruption of neurovascular coupling after CSD; the rise in CMRO(2) was unchanged. Our data suggest that blockade of mPTP formation and CaN activation may prevent persistent CBF reduction and vascular dysfunction after CSD.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Brain; Brain Injuries; Cerebrovascular Circulation; Cortical Spreading Depression; Cyclosporine; Enzyme Inhibitors; Immunosuppressive Agents; Male; Oxygen; Rats; Rats, Wistar; Tacrolimus; Vasoconstrictor Agents

Tropisetron is widely used to counteract chemotherapy-induced emesis. Evidence obtained from human and animal studies shows that tropisetron possesses anti-inflammatory properties. In this study, we assessed the effect of tropisetron on brain damage in a rat thromboembolic model of stroke. Stroke was rendered in rats by introduction of an autologous clot into the middle cerebral artery (MCA). Tropisetron (1 or 3mg/kg); m-chlorophenylbiguanide (mCPBG), a selective 5-HT(3) receptor agonist (15 mg/kg); tropisetron (3mg/kg) plus mCPBG (15 mg/kg); granisetron (3mg/kg); tacrolimus (1mg/kg); or tacrolimus (1mg/kg) plus tropisetron (3mg/kg) were administered intraperitoneally 1h prior to embolization. Behavioral scores and infarct volume as well as myeloperoxidase (MPO) activity and tumor necrosis factor-alpha (TNF-α) level were determined in the ipsilateral cortex 4h and 48 h following stroke induction. Forty-eight hours after embolization, tropisetron (1 or 3mg/kg), tropisetron (3mg/kg) plus mCPBG (15 mg/kg), tacrolimus (1mg/kg), or tacrolimus (1mg/kg) plus tropisetron (3mg/kg) significantly curtailed brain infarction, improved behavioral scores, diminished elevated tissue MPO activity, and reduced TNF-α levels compared to control group (n=6; P<0.05). mCPBG or granisetron had no effect on the mentioned parameters. Tropisetron attenuates brain damage after a thromboembolic event. Beneficial effects of tropisetron in this setting are receptor independent.

Topics: Analysis of Variance; Animals; Biguanides; Blood Gas Analysis; Brain Edema; Brain Infarction; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Immunosuppressive Agents; Indoles; Ischemia; Male; Nervous System Diseases; Peroxidase; Rats; Rats, Wistar; Seizures; Serotonin Antagonists; Stroke; Tacrolimus; Tropisetron; Tumor Necrosis Factor-alpha

Following traumatic brain injury (TBI), inhibition of reactive oxygen species and/or calcineurin can exert axonal and vascular protection. This protection proves optimal when these strategies are used early post-injury. Recent work has shown that the combination of delayed drug administration and delayed hypothermia extends this protection. Here we revisit this issue in TBI using the nitroxide antioxidant Tempol, or the immunophilin ligand FK506, together with delayed hypothermia, to determine their effects upon cerebral vascular reactivity and axonal damage. Animals were subjected to TBI and treated with Tempol at 30 or 90 min post-injury, or 90 min post-injury with concomitant mild hypothermia (33°C). Another group of animals were treated in the same fashion with the exception that they received FK506. Cranial windows were placed to assess vascular reactivity over 6 h post-injury, when the animals were assessed for traumatically induced axonal damage. Vasoreactivity was preserved by early Tempol administration; however, this benefit declined with time. The coupling of hypothermia and delayed Tempol, however, exerted significant vascular protection. The use of early and delayed FK506 provided significant vascular protection which was not augmented by hypothermia. The early administration of Tempol provided dramatic axonal protection that was not enhanced with hypothermia. Early and delayed FK506 provided significant axonal protection, although this protection was not enhanced by delayed hypothermia. The current investigation supports the premise that Tempol coupled with hypothermia extends its benefits. While FK506 proved efficacious with early and delayed administration, it did not provide either increased vascular or axonal benefit with hypothermia. These studies illustrate the potential benefits of Tempol coupled to delayed hypothermia. However, these findings do not transfer to the use of FK506, which in previous studies proved beneficial when coupled with hypothermia. These divergent results may be a reflection of the different animal models used and/or their associated injury severity.

Topics: Animals; Axons; Brain Injuries; Combined Modality Therapy; Cyclic N-Oxides; Disease Models, Animal; Hypothermia, Induced; Immunosuppressive Agents; Male; Microcirculation; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Spin Labels; Tacrolimus

Tacrolimus (FK506) and cyclosporin A (CsA), immunosuppressants widely used in post-transplantional therapy, have been reported to protect neurons in the injured brain. This effect can be exerted directly and indirectly via inflammatory cells. Since the data come exclusively from studies on the adult brain, we examined effects of the drugs on the macrophage recruitment in the brain injured at early developmental stages.. Following the brain injury, 1- and 6-day-old Wistar rats (P1s and P6s, respectively) were treated with FK506 or CsA and injected with [(3)H]thymidine. Brain sections were processed for BSI-B4 isolectin histochemistry and subjected to autoradiography to visualize proliferating and non-proliferating macrophages.. In P1s (n=33), FK506 evoked a dose-dependent reduction in the number of macrophages. P6s (n=30) presented greater decreases in macrophage numbers and their proliferative activity than the newborns. CsA application in P1s (n=27) affected neither recruitment of macrophages to the region of injury nor their proliferation. In CsA-treated P6s (n=28), reduction of the macrophage population and its proliferative activity was also seen but was much smaller than that following FK506 administration.. High effectiveness of FK506 in regulation of the inflammatory response and neuroprotection observed in the adult brain can also be considered as a possible indirect determinant of neuronal survival following the brain injury at very early developmental stages.

Topics: Age Factors; Animals; Animals, Newborn; Autoradiography; Brain; Brain Injuries; Cell Count; Cell Proliferation; Cyclosporine; Dose-Response Relationship, Drug; Immunosuppressive Agents; Inflammation; Macrophage Activation; Macrophages; Male; Neurons; Rats; Rats, Wistar; Statistics, Nonparametric; Tacrolimus

The expression of the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2/CXCL2) and the monocyte chemokine monocyte chemotactic protein-1 (MCP-1/CCL2) have been described in glial cells in vitro but their origin following TBI has not been established. Furthermore, little is known of the modulation of these chemokines. Chemokine expression was investigated in male Sprague-Dawley rats following moderate lateral fluid percussion injury (LFPI). At 0, 4, 8, 12, and 24 h after injury, brains were harvested and MIP-2/CXCL2 and MCP-1/CCL2 levels measured by ELISA. To investigate the inhibition of chemokine expression a second cohort of animals received dexamethasone (1-15mg/kg), FK506 (1mg/kg), or vehicle, systemically, immediately after injury. These animals were sacrificed at the time of peak chemokine expression. A third cohort of animals was also sacrificed at the time of peak chemokine expression and immunohistochemistry performed for MIP-2/CXCL2 and MCP-1/CCL2. Following LFPI, chemokines were increased in the ipsilateral hemisphere, MIP-2/CXCL2 peaking at 4 h and MCP-1/CCL2 peaking at 8-12 h post-injury. Dexamethasone significantly reduced cortical MCP-1/CCL2, but not MIP-2/CXCL2 concentrations. FK506 did not inhibit chemokine expression. In undamaged brain, chemokine expression was localized to cells with a neuronal morphology. For MIP-2/CXCL2 this was supported by double staining for the neuronal antigen NeuN. In contused tissue, increased MIP-2/CXCL2 and MCP-1/CCL2 staining was visible in cells with the morphology of degenerating neurons. MIP-2/CXCL2 and MCP-1/CCL2 are increased after injury, and neurons appear to be the source of this expression. Chemokine expression was selectively inhibited by dexamethasone. The implications of this are discussed.

Topics: Animals; Anti-Inflammatory Agents; Brain; Brain Injuries; Chemokine CCL2; Chemokine CXCL2; Dexamethasone; Disease Models, Animal; DNA-Binding Proteins; Down-Regulation; Immunosuppressive Agents; Male; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors; Up-Regulation

Prior investigations of traumatic axonal injury (TAI), and pharmacological treatments of TAI pathology, have focused exclusively on the role of myelinated axons, with no systematic observations directed towards unmyelinated axon pathophysiology. Recent electrophysiological evidence, however, indicates that unmyelinated axons are more vulnerable than myelinated axons in a rodent model of experimental TAI. Given their susceptibility to TAI, the present study examines whether unmyelinated axons also respond differentially to FK506, an immunophilin ligand with well-established neuroprotective efficacy in the myelinated fiber population. Adult rats received 3.0 mg/kg FK506 intravenously at 30 min prior to midline fluid percussion injury. In brain slice electrophysiological recordings, conducted at 24 h postinjury, compound action potentials (CAPs) were evoked in the corpus callosum, and injury effects quantified separately for CAP waveform components generated by myelinated axons (N1 wave) and unmyelinated axons (N2 wave). The amplitudes of both CAP components were suppressed postinjury, although this deficit was 16% greater for the N2 CAP. While FK506 treatment provided significant neuroprotection for both N1 and N2 CAPs, the drug benefit for the N2 CAP amplitude was 122% greater than that for the N1 CAPs, and improved postinjury strength-duration and refractoriness properties only in N2 CAPs. Immunocytochemical observations, of TAI reflected in intra-axonal pooling of amyloid precursor protein, indicated that FK506 reduced the extent of postinjury impairments to axonal transport and subsequent axonal damage. Collectively, these studies further substantiate a distinctive role of unmyelinated axons in TAI, and suggest a highly efficacious neuroprotective strategy to target this axonal population.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Brain Injuries; Corpus Callosum; Disease Models, Animal; Dose-Response Relationship, Radiation; Electric Stimulation; Evoked Potentials; In Vitro Techniques; Male; Multivariate Analysis; Nerve Fibers, Unmyelinated; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

The immunosuppressant drug tacrolimus (FK-506) failed to show an anti-edematous effect despite suppressing pro-inflammatory cytokines in cerebrospinal fluid following focal traumatic brain injury. By questioning the role of the inflammatory response as a pharmacological target, we investigated the effects of FK-506 on immune cell infiltration in brain-injured rats. Following induction of a cortical contusion, male Sprague-Dawley rats received FK-506 or physiological saline intraperitoneally. Brains were removed at 24 h, 72 h or 7 days, respectively. Frozen brain sections (7 microm) were stained immunohistologically for markers of endothelial activation (intercellular adhesion molecule-1--ICAM-1), neutrophil infiltration (His-48), and microglial and macrophage activation (Ox-6; ED-1), respectively. Immunopositive cells were counted microscopically. Contusion volume (CV) was quantified morphometrically 7 days after trauma. Inflammatory response was confined to the ipsilateral cortex and hippocampal formation, predominating in the contusion and pericontusional cortex. Strongest ICAM-1 expression coincided with sustained granulocyte accumulation at 72h which was suppressed by FK-506. Ox-6+ cells prevailing at 72 h were also significantly reduced by FK-506. ED-1+ cells reaching highest intensity at 7 days were significantly attenuated at 72 h. Cortical CV was not influenced. FK-506 significantly decreased post-traumatic local inflammation which, however, was not associated with a reduction in cortical CV. These results question the importance of post-traumatic local immune cell infiltration in the secondary growth of a cortical contusion.

Topics: Animals; Brain; Brain Injuries; Immunosuppressive Agents; Intercellular Adhesion Molecule-1; Macrophage Activation; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Tacrolimus

Susceptibility of the injured rat brain to seizures depends on the developmental stage at which the injury had been inflicted. Our previous study shows that tacrolimus (FK506) and cyclosporin A (CsA) applied following the injury can also decrease or increase the seizure susceptibility in an age-dependent way. To find possible neuronal substrates of the effects, we examined influences of the agents on the injured brain and on its neuronal population. Rat brains were mechanically injured on postnatal days 6 (P6) or 30 (P30). Twenty minutes and 24 hours following the injury, FK506 or CsA were injected in clinically used pharmaceutical formulations (Prograf or Sandimmun, respectively). The brains were fixed on postnatal day 60 and processed for histological examinations. To detect if negative effects of the injury could be abolished by the treatments, we examined the brain weight, the size of the injured region, and the nerve cell density, including the density of calretinin- and parvalbumin-immunopositive cells. We have found that long-term effects of treatments with the FK506- and CsA-containing pharmaceutical formulations were never better than those of the vehicle alone (Cremophor and ethanol mixture). Moreover, the treatments could even amplify negative consequences of the injury alone. It could, therefore, be concluded that all the neuroprotective effects observed in the present study resulted exclusively from the influence of the vehicle alone. These effects of the brain injury and of subsequent treatments performed at different developmental stages were considered as possible determinants of further increase or decrease in susceptibility to seizures observed in adulthood.

Topics: Animals; Brain; Brain Injuries; Calbindin 2; Cell Count; Chemistry, Pharmaceutical; Cyclosporine; Epilepsy; Female; Immunosuppressive Agents; Neurons; Neuroprotective Agents; Organ Size; Parvalbumins; Pregnancy; Rats; Rats, Wistar; S100 Calcium Binding Protein G; Staining and Labeling; Tacrolimus

Traumatic axonal injury (TAI) following traumatic brain injury (TBI) remains a clinical problem for which no effective treatment exists. TAI was thought to involve intraaxonal changes that universally led to impaired axonal transport (IAT), disconnection and axonal bulb formation. However, recent, immunocytochemical studies employing antibodies to amyloid precursor protein (APP), a marker of IAT and antibodies to neurofilament compaction (NFC), RM014, demonstrated that NFC typically occurs independent of IAT, indicating the existence of different populations of damaged axons. FK506 administration has been shown to attenuate IAT. However, in light of the above, the ability of FK506 to attenuate axonal damage demonstrating NFC requires evaluation. The current study explored the potential of FK506 to attenuate both populations of damaged axons. Rats were administered FK506 (3 mg/kg) or vehicle 30 min preinjury. Three hours post-TBI, tissue was prepared for the visualization of TAI using antibodies targeting IAT (APP) or NFC (RMO14) or a combined labeling strategy. Confirming previous reports, FK506 treatment reduced the number of axons demonstrating IAT in the CSpT, from 411 +/- 54.70 to 91.00 +/- 33.87 (P

Topics: Amyloid beta-Protein Precursor; Animals; Axonal Transport; Brain Injuries; Diffuse Axonal Injury; Disease Models, Animal; Immunohistochemistry; Immunosuppressive Agents; Male; Neurofilament Proteins; Neurologic Examination; Pyramidal Tracts; Random Allocation; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors

Neuronal and glial cell death caused by axonal injury sometimes contributes to whole brain pathology after traumatic brain injury (TBI). We show that neuroprotection by 2 types of immunosuppressants, cyclosporin A (CsA) and tacrolimus (FK506), in a cryogenic brain injury model results from inhibition of calcineurin and protection from mitochondrial damage caused by formation of a mitochondrial permeability transition pore induced by cyclophilin D (CyPD), one of the prolyl cis/trans isomerase family members. We evaluated why CsA is neuroprotective by microarray analysis of gene expression in the cryogenic brain injury rat model. Analyses of expression patterns demonstrated that expression of over 14,000 genes changed between the groups with and without CsA treatment, and about 350 genes among them were extracted showing a significant difference. We learned that the differential expression of several gene targets showed specific patterns in a time-dependent manner. These results may help elucidate the mechanisms of neuronal cell death after TBI and the neuroprotective effects of CsA after TBI.

Topics: Animals; Biomarkers; Brain; Brain Injuries; Cyclosporine; Gene Expression Profiling; Gene Expression Regulation; Genetic Markers; Male; Nerve Tissue Proteins; Neuroprotective Agents; Oligonucleotide Array Sequence Analysis; Prognosis; Rats; Rats, Wistar; Tacrolimus; Time Factors; Treatment Outcome

Brain injury is often associated with proliferation and hypertrophic response of glial cells (reactive gliosis). We have previously reported immunosuppressant effects on survival of glioma cells and adult reactive astrocytes. In the present study, we demonstrate growth-inhibitory effect of FK506 on cortical astrocytes from newborn rats. FK506 inhibits Erk and PI-3K/Akt signaling, two crucial pro-survival pathways. The levels of phosphorylated Akt and p42/44 Erk decline in few hours after FK506 addition. Furthermore, in FK506-treated astrocyte cultures the levels of mRNA encoding PDGF, bFGF, and CNTF decreased. Downregulation of growth factor expression by FK506 may play a role in the inhibition of mitogenic/hypertrophic responses. FasL mRNA level was elevated and interaction of FasL with Fas receptor expressed in astrocytes may trigger cell death. Interestingly, expression of BDNF increased in a dose-dependent manner in FK506-treated astrocytes. Upregulation of BDNF mRNA and protein level in astrocytes exposed to FK506 may underlie neuroprotective action of FK506.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Brain-Derived Neurotrophic Factor; Cell Survival; Cells, Cultured; Cicatrix; Dose-Response Relationship, Drug; Down-Regulation; Fas Ligand Protein; Gene Expression Regulation; Gliosis; Growth Substances; Immunosuppressive Agents; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Neuroprotective Agents; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Tacrolimus; Up-Regulation

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.

Topics: Animals; Astrocytes; Brain Injuries; Calcineurin Inhibitors; Cells, Cultured; Cyclosporine; Free Radicals; Intracellular Membranes; Microglia; Mitochondria; Neurons; Permeability; Rats; Reference Values; Tacrolimus; Time Factors

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Astrocytes; Brain; Brain Injuries; Brain Ischemia; Cell Survival; Functional Laterality; Glial Fibrillary Acidic Protein; HSP70 Heat-Shock Proteins; Immunohistochemistry; Immunosuppressive Agents; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Rats; Rats, Inbred SHR; Tacrolimus; Vimentin

Disturbance of calcium homeostasis contributes to evolving tissue damage and energetic impairment following traumatic brain injury (TBI). Calcium-mediated activation of calcineurin results in production of tissue-damaging nitric oxide and free oxygen radicals. Inhibition of calcineurin induced by the immunosuppressant tacrolimus (FK506) has been shown to reduce structural and functional damage after ischemia. The aims of the present study were to investigate time- and dose-dependent short-term antiedematous effects of tacrolimus following TBI.. A left temporoparietal contusion (controlled cortical impact injury [CCII]) was induced in 51 male Sprague-Dawley rats. Tacrolimus (1 or 3 mg/kg body weight) was administered by a single intraperitoneal injection at 5 minutes, 30 minutes, or 4 hours after CCII occurred. Control rats received physiological saline. Water contents of traumatized and nontraumatized hemispheres, as well as cerebrospinal fluid (CSF) levels of mediators reflecting tissue damage (the proinflammatory cytokines interleukin [IL]-6 and tumor necrosis factor [TNF]-alpha, the excitotoxin glutamate, and the adenosine triphosphate-degradation product hypoxanthine), were determined 24 hours after trauma. Although CSF levels of IL-6 and TNFalpha were completely suppressed by tacrolimus at all time points and at both concentrations, CSF levels of glutamate and hypoxanthine, as well as edema formation, were only marginally influenced. Significant reduction of cerebral water content was confined to nontraumatized hemispheres. In addition, the higher dose of tacrolimus failed to exert significant antiedematous effects on traumatized hemispheres.. Under the present study design, the potency of tacrolimus in reducing edema formation following CCII seems limited. However, its immunosuppressive effects could be of value in influencing the posttraumatic inflammatory response known to aggravate tissue damage.

Topics: Animals; Brain Edema; Brain Injuries; Calcineurin; Cerebral Cortex; Energy Metabolism; Glutamic Acid; Hypoxanthine; Immunosuppressive Agents; Interleukin-6; Male; Rats; Rats, Sprague-Dawley; Tacrolimus; Tumor Necrosis Factor-alpha; Water

Immunosuppressant drugs, like FK506, and nonimmunosuppressant compounds like, GPI1046 and L685818, are immunophilin ligands that specifically bind to immunophilins, like FK506 binding protein 12 (FKBP12). Several lines of evidence show that these ligands exert neurotrophic properties in neural injury models and in PC12 cells. However, the mechanism of the neurotrophic function of the immunophilin ligands is poorly known. In the present study, we use MPP+ and 6-OHDA toxicity models to examine both neuroprotective and neuroregenerative effects of immunophilin ligands on primary cultures of midbrain dopaminergic neurons. We find that FK506, GPI1046 and L685818 at concentrations from 0.01 to 1 microM partially, but significantly, protect dopaminergic neurons against both MPP+ and 6-OHDA toxicity. By Western blot analysis, we also find that all three compounds prevent tyrosine hydroxylase (TH) loss induced by MPP+ and 6-OHDA treatments. Morphologic analysis of dopaminergic neurons, by immunocytochemistry, shows that MPP+ and 6-OHDA cause the retraction and loss of neuronal processes, while FK506, GPI1046 and L685818 promote regeneration of these processes as indicated by increases in process number and length. To examine if FKBP12 is required for neurotrophic effects of immunophilin ligands, we cultured dopaminergic neurons from FKBP12 knockout mice and find that FK506 still protects dopaminergic neurons against MPP+ toxicity. These results suggest that FKBP12 is not essential for the neurotrophic properties of immunophilin ligands, and immunophilin ligands are a new class of neuroprotective and neuroregenerative agents that may have therapeutic potential in a variety of neurological disorders.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Brain Injuries; Cells, Cultured; Dopamine; Immunophilins; Immunosuppressive Agents; Ligands; Mesencephalon; Mice; Mice, Knockout; Nerve Degeneration; Nerve Regeneration; Neurons; Neuroprotective Agents; Neurotoxins; Oxidopamine; Parkinsonian Disorders; Pyrrolidines; Rats; Rats, Sprague-Dawley; Tacrolimus; Tacrolimus Binding Protein 1A; Tyrosine 3-Monooxygenase

The immunophilin ligand, cyclosporin A (CsA), is effective in reducing the axonal damage associated with traumatic brain injury (TBI). Based upon extensive ultrastructural and immunohistochemical studies, the neuroprotection afforded by CsA appeared to be mediated via mitochondrial protection, specifically, the prevention of mitochondrial swelling and inhibition of mitochondrial permeability transition (MPT). However, the potential that CsA could also be neuroprotective via the immunophilin-mediated inhibition of the protein phosphatase, calcineurin (CN) has not been directly assessed. To address this issue, the current study assessed the ability of FK506, another immunophilin ligand that inhibits CN with no effect on MPT, to attenuate axonal damage in a rat impact-acceleration model of TBI. Traumatic axonal injury (TAI), detected via an antibody against beta-amyloid precursor protein (APP), a specific marker of axonal injury, was significantly reduced at 24 hr postinjury in Sprague-Dawley rats receiving intravenous FK506 (2 mg/kg; n = 5) 30 min prior to injury compared to vehicle controls (n = 3). While not rejecting the established efficacy of CsA in providing neuroprotection via its targeting of MPT, this study does underscore the potential importance of CN in the progressive pathobiology of TAI, suggesting that CN may constitute another important therapeutic target.

Topics: Acceleration; Animals; Axons; Blood Gas Analysis; Brain; Brain Injuries; Image Processing, Computer-Assisted; Immunohistochemistry; Immunosuppressive Agents; Injections, Intravenous; Male; Rats; Rats, Sprague-Dawley; Tacrolimus

Our laboratory has shown that traumatically induced axonal injury (TAI) is significantly reduced by posttraumatic hypothermia followed by slow rewarming. Further, TAI can be exacerbated by rapid rewarming, and the damaging consequences of rapid rewarming can be reversed by cyclosporin A, which is believed to protect via blunting mitochondrial permeability transition (MPT). In this communication, we continue investigating the damaging consequences of rapid posthypothermic rewarming and the protective role of immunophilin ligands using another member of the immunophilin family, FK506, which does not affect MPT but rather inhibits calcineurin. Rats were subjected to impact-acceleration brain injury followed by the induction of hypothermia with subsequent rapid or slow posthypothermic rewarming. During rewarming, animals received either FK506 or its vehicle. Three hours postinjury, animals were prepared for the visualization of TAI via antibodies targeting impaired axoplasmic transport (APP) and/or overt neurofilament alteration (RMO-14). Rapid rewarming exacerbated TAI, which was attenuated by FK506. This protection was statistically significant for the APP-immunoreactive fibers but not for the RMO-14-positive fibers. Combined labeling, using one chromagen to visualize both axonal changes, suggested that these two immunoreactive profiles revealed two distinct pathologies not occurring along the same axon. Collectively, these studies confirmed previous observations identifying the adverse consequences of rapid rewarming while also showing the complexity of the pathobiology of TAI. Additionally, the demonstration that FK506 is protective suggests that calcineurin may be a major target for neuroprotection.

Topics: Animals; Axonal Transport; Axons; Blood Pressure; Body Temperature; Brain Injuries; Calcineurin Inhibitors; Cell Count; Hypothermia, Induced; Immunohistochemistry; Immunophilins; Immunosuppressive Agents; Ligands; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Rewarming; Tacrolimus; Time Factors

Cyclosporin A (CsA) reduces ischemic brain damage when administered in such a way that its penetration across the blood-brain barrier is enhanced. Since only pretreatment has previously been used in focal ischemia, the objective of the present study was to establish whether posttreatment is efficacious and to assess the window of therapeutic opportunity for CsA. To that end, CsA was given 5 min to 6 h after the start of reperfusion following 2 h transient ischemia, and infarct volume was assessed after 48 h by triphenyltetrazolium chloride staining. Attempts were made to circumvent the BBB to CsA by an intracerebral needle lesion, by an increase in the intravenous CsA dose, or by osmotic opening with intracarotid mannitol. The results were compared to those obtained with FK506. Intravenous CsA in a dose of 10 mg/kg failed to reduce infarct volume, unless preceded by a needle lesion. That procedure, and an increase in CsA dose to 50 mg/kg, reduced infarct volume to about 50% of control, but the higher dose had toxic side effects. The coupled intracarotid infusion of mannitol and CsA (10 mg/kg) was more efficacious, without overt side effects. However, mannitol proved dispensable since CsA alone reduced infarct volume to 30% of control, with a therapeutic window of 3-6 h. When given after 5 min of reflow, CsA reduced infarct volume to 10% of control and was clearly more neuroprotective than FK506. Possibly, this is because CsA blocks the mitochondrial permeability transition pore which is opened under adverse conditions.

Topics: Animals; Blood Glucose; Blood-Brain Barrier; Body Temperature; Brain Injuries; Carbon Dioxide; Carotid Arteries; Cyclosporine; Diuretics, Osmotic; Hydrogen-Ion Concentration; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Injections, Intra-Arterial; Ischemic Attack, Transient; Male; Mannitol; Osmosis; Oxygen; Rats; Rats, Wistar; Sodium Chloride; Tacrolimus; Wounds, Stab