Nadezda Lukacova

Peripheral axotomy affects nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthases in the spinal cord of the rabbit

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium‐dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS‐IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS‐IR appeared in a large number of small and medium‐sized to large neurons in the ipsilateral L4–L6 DRG, accompanied by enhanced NOS‐IR of thick myelinated fibers in the ipsilateral L4–L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I–IV in the ipsilateral medial part of the dorsal horn in L4–L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd‐exhibiting neurons in laminae I–II and, in contrast to this, a statistically significant increase of medium‐sized to large NADPHd‐stained somata in the ipsilateral laminae III–VI of L4–L6 segments were found. A detailed compartmentalization of L4–L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd‐exhibiting and NOS‐IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo‐synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS‐IR in the ipsilateral dorsal roots and dorsal horn of L4–L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.

The Case for the Bulbospinal Respiratory Nitric Oxide Synthase-Immunoreactive Pathway in the Dog

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3-C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 microm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2-C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

Suppression of stretch reflex activity after spinal or systemic treatment with AMPA receptor antagonist NGX424 in rats with developed baclofen tolerance.

BACKGROUND AND PURPOSE Baclofen (a GABAB receptor agonist) is the most commonly used anti‐spasticity agent in clinical practice. While effective when administered spinally or systemically, the development of progressive tolerance represents a serious limitation for its long‐term use. The goal of the present study was to characterize the treatment potency after intrathecal or systemic treatment with the selective AMPA receptor antagonist NGX424 on stretch reflex activity (SRA) and background muscle activity (BMA) in rats with developed baclofen tolerance.

Localization and Distribution Patterns of Nicotinamide Adenine Dinucleotide Phosphate Diaphorase Exhibiting Axons in the White Matter of the Spinal Cord of the Rabbit

The funicular distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting axons was examined in the white matter of the rabbit spinal cord by using horizontal, parasaggital, and transverse sections. Four morphologically distinct kinds of NADPHd-exhibiting axons (2.5–3.5 μm in diameter) were identified in the sulcomarginal fasciculus as a part of the ventral column in the cervical and upper thoracic segments and in the long propriospinal bundle of the ventral column in Th3–L3 segments. Varicose NADPHd-exhibiting axons of the sympathetic preganglionic neurons, characterized by widely spaced varicosities, were found in the ventral column of Th2–L3 segments. A third kind of NADPHd-positive ultrafine axons, 0.3–0.5 μm in diameter with numerous varicosities mostly spherical in shape, was identified in large number within Lissauers tract. The last group of NADPHd-exhibiting axons (1.0–1.5 μm in diameter) occurred in the Lissauer tract. Most of these axons were traceable for considerable distances and generated varicosities varying in shape from spherical to elliptical forms. The majority of NADPHd-exhibiting axons identified in the cuneate and gracile fascicles were concentrated in the deep portion of the dorsal column. An extremely reduced number of NADPHd-exhibiting axons, confirmed by a computer-assisted image-processing system, was found in the dorsal half of the gracile fascicle. Axonal NADPHd positivity could not be detected in a wide area of the lateral column consistent with the location of the dorsal spinocerebellar tract. Numerous, mostly thin NADPHd-positive axonal profiles were detected in the dorsolateral funiculus in all the segments studied and in a juxtagriseal portion of the lateral column as far as the cervical and lumbar enlargements. A massive occurrence of axonal NADPHd positivity was detected in the juxtagriseal layer of the ventral column all along the rostrocaudal axis of the spinal cord. The prominent NADPHd-exhibiting bundles containing thick, smooth, nonvaricose axons were identified in the mediobasal and central portion of the ventral column. First, the sulcomarginal fasciculus was found in the basal and medial portion of the ventral column in all cervical and upper thoracic segments. Second, more caudally, a long propriospinal bundle displaying prominent NADPHd positivity was localized in the central portion of the ventral column throughout the Th3–L3 segments.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: Effect on spinally grafted human neural precursor survival

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 ± 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

Effect of Midthoracic Spinal Cord Constriction on Catalytic Nitric Oxide Synthase Activity in the White Matter Columns of Rabbit

The distribution and changes of catalytic nitric oxid synthase (cNOS) activity in the dorsal, lateral and ventral white matter columns at midthoracic level of the rabbits spinal cord were studied in a model of surgically-induced spinal cord constriction performed at Th7 segment level and compared with the occurrence of nicotinamide adenine dinucleotide phosphate diaphorase expressing and neuronal nitric oxide synthase immunoreactive axons in the white matter of the control thoracic segments. Segmental and white-column dependent differences of cNOS activity were found in the dorsal (141.5 ± 4.2 dpm/μm protein), lateral (87.3 ± 11.5 dpm/μm protein) and ventral (117.1 ± 7.6 dpm/μm protein) white matter columns in the Th5-Th6 segments and in the dorsal (103.3 ± 15.5 dpm/μm protein), lateral (54.9 ± 4.9 dpm/μm protein), and ventral (86.1 ± 6.8 dpm/μm protein) white matter columns in the Th8-Th9 segments. A surgically-induced constriction of Th7 segment caused a disproportionate response of cNOS activity in the rostrally (Th5-Th6) and caudally (Th8-Th9) located segments in both lateral and ventral white matter columns. While a statistically significant decrease of cNOS activity was detected above the constriction site in the ventral columns, a considerable, statistically significant increase of cNOS activity was noted in the white lateral columns below the site of constriction. It is reasoned that the changes of cNOS activity may have adverse effects on nitric oxide (NO) production in the white matter close to the site of constriction injury, thus broadening the scope of the secondary mechanisms that play a role in neuronal trauma.

Potent suppression of stretch reflex activity after systemic or spinal delivery of tizanidine in rats with spinal ischemia-induced chronic spastic paraplegia

BACKGROUND Spasticity and rigidity are serious complications associated with spinal traumatic or ischemic injury. Clinical studies show that tizanidine (Tiz) is an effective antispasticity agent; however, the mechanism of this effect is still not clear. Tiz binds not only to α2-adrenoreceptors (AR) but also to imidazoline (I) receptors. Both receptor systems (AR+I) are present in the spinal cord interneurons and α-motoneurons. The aim of the present study was to evaluate the therapeutic potency of systematically or spinally (intrathecally [IT]) delivered Tiz on stretch reflex activity (SRA) in animals with ischemic spasticity, and to delineate supraspinal or spinal sites of Tiz action. EXPERIMENTAL PROCEDURES Animals were exposed to 10 min of spinal ischemia to induce an increase in SRA. Increase in SRA was identified by simultaneous increase in recorded electromyography (EMG) activity and ankle resistance measured during computer-controlled ankle dorsiflexion (40°/3 s) in fully awake animals. Animals with increased SRA were divided into several experimental subgroups and treated as follows: (i) Tiz administered systemically at the dose of 1 mg kg(-1), or IT at 10 μg or 50 μg delivered as a single dose; (ii) treatment with systemic Tiz was followed by the systemic injection of vehicle, or by nonselective AR antagonist without affinity for I receptors; yohimbine (Yoh), α2A AR antagonist; BRL44408 (BRL), α2B AR antagonist; ARC239 (ARC), nonselective AR and I(1) receptor antagonist; efaroxan (Efa), or nonselective AR and I(2) receptor antagonist; idazoxan (Ida); (iii) treatment with IT Tiz was followed by the IT injection of selective α2A AR antagonist; atipamezole (Ati). In a separate group of spastic animals the effect of systemic Tiz treatment (1 mg/kg) or isoflurane anesthesia on H-reflex activity was also studied. RESULTS Systemic and/or IT treatment with Tiz significantly suppressed SRA. This Tiz-mediated anti-SRA effect was reversed by BRL (5 mg kg(-1)), Efa (1 mg kg(-1)), and Ida (1 mg kg(-1)). No reversal was seen after Yoh (3 mg kg(-1)) or ARC (5 mg kg(-1)) treatment. Anti-SRA induced by IT Tiz (50 μg) was reversed by IT injection of Ati (50 μg). Significant suppression of H-reflex was measured after systemic Tiz treatment (1 mg/kg) or isoflurane (2%) anesthesia, respectively. Immunofluorescence staining of spinal cord sections taken from animals with spasticity showed upregulation of α2A receptor in activated astrocytes. CONCLUSIONS These data suggest that α2A AR and I receptors, but not α2B AR, primarily mediate the Tiz-induced antispasticity effect. This effect involves spinal and potentially supraspinal sites and likely targets α2A receptor present on spinal neurons, primary afferents, and activated astrocytes. Further studies using highly selective antagonists are needed to elucidate the involvement of specific subtypes of the AR and I receptors in the antispasticity effect seen after Tiz treatment.

Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization.

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.

Regional distribution of phospholipids and polyphosphatidyl inositides in the rabbit's spinal cord.

The plasticity of the membrane phospholipids in general and stimulated phosphoinositides turnover in particular are the subjects in a variety of neural paradigms studying the molecular mechanisms of neuronal changes under normal and pathological conditions. The regional modifiability of phospholipids (SM, PC, PS, PI, PA + DG, PE), polyphosphatidylinositides (PI, PIP, PIP2) and diacylglycerol-dependent incorporation of CDP-choline into phosphatidylcholine in the gray matter, white matter, dorsal horns, intermediate zone and ventral horns of the rabbits spinal cord was studied. We have found 1. a significant increase in the concentration of SM, PC, PS, DG + PA and PE in the white matter in comparison to the gray one, 2. the highest concentration of the outer membrane leaflet-bound phospholipids in the dorsal horns and the inner membrane phospholipids in the intermediate zone in comparison to the gray matter, 3. a substantial amount of labeled polyphosphatidylinositides (poly-PIs) in the spinal cord white matter with descending order PIP > PI > PIP2, 4. similar incorporation of myo-2-[3H]inositol into all poly-PIs in ventral horns and intermediate zone, but a different, lower incorporation into PI and PIP and higher into PIP2 in the dorsal horns, 5. higher diacylglycerol-dependent incorporation of CDP-choline into PC in the regionally undivided gray matter than in the white matter taken as a whole, 6. the high proportion of diacylglycerol-dependent incorporation of CDP-choline into PC in both the ventral and dorsal horns, whereas that in the intermediate zone remained low.

A Single Dose of Atorvastatin Applied Acutely after Spinal Cord Injury Suppresses Inflammation, Apoptosis, and Promotes Axon Outgrowth, Which Might Be Essential for Favorable Functional Outcome

The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1β release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.