Immunosuppressant FK506: focusing on neuroprotective effects following brain and spinal cord injury.

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.

Effects of short-duration electromagnetic radiation on early postnatal neurogenesis in rats: Fos and NADPH-d histochemical studies.

The immediate effects of whole body electromagnetic radiation (EMR) were used to study postnatal neurogenesis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Wistar rats of both sexes. Newborn postnatal day 7 (P7) and young adult rats (P28) were exposed to pulsed electromagnetic fields (EMF) at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm(2) for 2 h. Post-irradiation changes were studied using immunohistochemical localization of Fos and NADPH-d. We found that short-duration exposure induces increased Fos immunoreactivity selectively in cells of the SVZ of P7 and P28 rats. There were no Fos positive cells visible within the RMS of irradiated rats. These findings indicate that some differences exist in prerequisites of proliferating cells between the SVZ and RMS regardless of the age of the rats. Short-duration exposure also caused praecox maturation of NADPH-d positive cells within the RMS of P7 rats. The NADPH-d positive cells appeared several days earlier than in age-matched controls, and their number and morphology showed characteristics of adult rats. On the other hand, in the young adult P28 rats, EMR induced morphological signs typical of early postnatal age. These findings indicate that EMR causes age-related changes in the production of nitric oxide (NO), which may lead to different courses of the proliferation cascade in newborn and young adult neurogenesis.

Effects of long-term FK506 administration on functional and histopathological outcome after spinal cord injury in adult rat.

FK506 (tacrolimus), a potent immunosuppressive drug primarily used for reduction of allograft rejection in organ transplantation, also offers neuroprotection after central nervous system injury. FK506-mediated immunosuppression and neuroprotection may occur through different mechanisms that could affect neurological recovery and the severity of spinal lesions where cells transplantation therapy is combined with FK506 application. We assessed effects of long-term FK506 administration using the same dose regiment (1 mg/kg/day for 6 weeks) as is used in spinal cord transplantation studies following a balloon-compression induced spinal cord injury (SCI). Body weight and locomotor recovery quantified by the BBB (Basso-Beattie-Bresnehan) locomotor rating scale were evaluated for up to 42 days post-injury. The area of the preserved spinal cord tissue within a 13 mm segment of the spinal cord (lesion epicenter and 6 mm rostral-caudal) was examined histologically. The results showed no significant effects of FK506 on spinal cord tissue sparing or improvement of locomotor recovery. However, body weight fell significantly (P < 0.05) with FK506 treatment when compared with placebo from day 7 until sacrifice. In our experimental design, long-term FK506 treatment did not affect the parameters of outcome following balloon-compression SCI in the rat; however, multiple effects of FK506 should be taken into account when evaluating the outcomes in transplantation studies.

Immunohistochemical study of postnatal neurogenesis after whole-body exposure to electromagnetic fields: evaluation of age- and dose-related changes in rats.

It is well established that strong electromagnetic fields (EMFs) can give rise to acute health effects, such as burns, which can be effectively prevented by respecting exposure guidelines and regulations. Current concerns are instead directed toward the possibility that long-term exposure to weak EMF might have detrimental health effects due to some biological mechanism, to date unknown. (1) The possible risk due to pulsed EMF at frequency 2.45 GHz and mean power density 2.8 mW/cm(2) on rat postnatal neurogenesis was studied in relation to the animal's age, duration of the exposure dose, and post-irradiation survival. (2) Proliferating cells marker, BrdU, was used to map age- and dose-related immunohistochemical changes within the rostral migratory stream (RMS) after whole-body exposure of newborn (P7) and senescent (24 months) rats. (3) Two dose-related exposure patterns were performed to clarify the cumulative effect of EMF: short-term exposure dose, 2 days irradiation (4 h/day), versus long-term exposure dose, 3 days irradiation (8 h/day), both followed by acute (24 h) and chronic (1-4 weeks) post-irradiation survival. (4) We found that the EMF induces significant age- and dose-dependent changes in proliferating cell numbers within the RMS. Our results indicate that the concerns about the possible risk of EMF generated in connection with production, transmission, distribution, and the use of electrical equipment and communication sets are justified at least with regard to early postnatal neurogenesis.

Limited minocycline neuroprotection after balloon-compression spinal cord injury in the rat.

Minocycline (MC), a second-generation tetracycline and anti-inflammatory agent reportedly provides neuroprotection following CNS injury. The objective of this study was to examine the neuroprotective effects of short and long-term MC treatment using balloon-compression spinal cord injury (SCI) in the rat. Rats subjected to SCI were treated with MC for 1 day (1DMC group; total dose 180 mg/kg) or 5 days (5DMC group; total dose 450 mg/kg) or placebo. The effects of MC treatment on locomotor recovery (BBB scale) and spinal cord white and gray matter sparing were evaluated for up to 28 days. Morphometric analysis showed that while MC treatment spared spinal cord white and gray matter rostral to the lesion epicenter in both, 1DMC and 5DMC groups, sparing of white and gray matter areas was not observed caudal to the traumatic lesion. In addition, MC treatment had no effect on final locomotor recovery. Limited improvement of spinal cord post-compression consequences raises questions about the neuroprotection efficiency of MC treatment following compression SCI in the rat.

Fluoro-Jade B staining following zymosan microinjection into the spinal cord white matter.

1. The fluorescein derivate Fluoro-Jade B (FJB), which primarily labels dead or dying neurons, was used to study the acute focal inflammation in the spinal cord white matter. Inflammation was induced by microinjection of the yeast particulate zymosan to evaluate the biological effects of intraspinal macrophages activation without the confounding effects of physical trauma. 2. A single bolus of zymosan (Sigma, 75 nL) was stereotaxically injected at the thoracic level into the lateral white matter of rat spinal cord. A standard Fluoro-Jade B staining protocol was applied to spinal cord sections at 6, 12, 24 h and 2, 4 days postinjection. Neutral Red, NADPH-diaphorase, Iba1-IR, and DAPI staining protocols accomplished examination of the cells participating in the acute inflammatory response. 3. Zymosan caused formation of clearly delineated inflammation lesions localized in the lateral white matter of the spinal cord. Fluoro-Jade B stained cells in the area of inflammation were not observed at 12 h postinjection while mild FJB staining appeared at 24 h and intense staining was observed at 2 and 4 days postinjection. 4. This study shows that the acute response to zymosan-induced inflammation in the rat spinal cord white matter causes a gradual appearance of phagocytic microglia/macrophages and delayed FJB staining of the inflammatory cells. 5. FJB, a reliable marker of dying neurons, is a more universal agent than formerly believed. One possible explanation for the gradual appearance of FJB-stained cells in the area of inflammation is that specific time is required for sufficient levels of proteins and/or myelin debris of axonal origin to appear in the cytoplasm of phagocytic microglia/macrophages.

Chronically implanted electrodes for repeated stimulation and recording of spinal cord potentials.

We have recorded and characterized the spinal cord evoked potentials (SCEPs) from the epidural space in the halothane-anesthetized rats. A group of 11 adult Wistar male rats was chronically implanted with two pairs of epidural electrodes. SCEPs were repeatedly elicited by applying electrical stimuli via bipolar U-shaped electrodes to the dorsal aspect of the spinal cord at C3-4 or Th11-12 levels, respectively. Responses were registered with the other pair of implanted electrodes, thus allowing us to monitor the descending (stimulation cervical/recording thoracic) and ascending SCEPs (stimulation thoracic/recording cervical). We studied the time-dependent changes of several SCEP parameters, among them the latency and amplitude of two major negative waves N1 and N2. During 4-weeks' survival, all major components of recordings remained stable and only minor changes in some parameters of the SCEPs were detected. We concluded that this technique enables repeated quantitative analysis of the conductivity of the spinal cord white matter in the rat. Our results indicate that SCEPs could be used in long-term experiments for monitoring progressive changes (degeneration/regeneration) in long projection tracts, primarily those occupying the dorsolateral quadrants of the spinal cord. These include projections that are of interest in spinal cord injury studies, i.e. ascending primary afferents, and important descending pathways including corticospinal, rubrospinal, reticulospinal, raphespinal and vestibulospinal tracts.