Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

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.

Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells.

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.

Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study.

Transient spinal cord ischemia may lead to a progressive degeneration of spinal interneurons and subsequently to increased hind limb motor tone. In the present work we sought to characterize the rigidity and spasticity components of this altered motor function by: i) tonic electromyographic activity measured in gastrocnemius muscle before and after ischemia, ii) measurement of muscle resistance during the period of ankle flexion and corresponding changes in electromyographic activity, iii) changes in Hoffmann reflex, and, iv) motor evoked potentials. In addition the effect of intrathecal treatment with baclofen (GABAB receptor agonist; 1 microg), nipecotic acid (GABA uptake inhibitor; 300 microg) and dorsal L2-L5 rhizotomy on spasticity and rigidity was studied. Finally, the changes in spinal choline acetyltransferase (ChAT) and vesicular glutamate transporter 2 and 1 (VGLUT2 and VGLUT1) expression were characterized using immunofluorescence and confocal microscopy. At 3-7 days after ischemia an increase in tonic electromyographic activity with a variable degree of rigidity was seen. In animals with modest rigidity a velocity-dependent increase in muscle resistance and corresponding appearance in electromyographic activity (consistent with the presence of spasticity) was measured during ankle rotation (4-612 degrees /s rotation). Measurement of the H-reflex revealed a significant increase in Hmax/Mmax ratio and a significant loss of rate-dependent inhibition. In the same animals a potent increase in motor evoked potential amplitudes was measured and this change correlated positively with the increased H-reflex responses. Spasticity and rigidity were consistently present for a minimum of 3 months after ischemia. Intrathecal treatment with baclofen (GABA B receptor agonist) and nipecotic acid (GABA uptake inhibitor) provided a significant suppression of spasticity, rigidity, H-reflex or motor evoked potentials. Dorsal L2-L5 rhizotomy significantly decreased muscle resistance but had no effect on increased amplitudes of motor evoked potentials. Confocal analysis of spinal cord sections at 8 weeks-12 months after ischemia revealed a continuing presence of ChAT positive alpha-motoneurons, Ia afferents and VGLUT2 and VGLUT1-positive terminals but a selective loss of small presumably inhibitory interneurons between laminae V-VII. These data demonstrate that brief transient spinal cord ischemia in rat leads to a consistent development of spasticity and rigidity. The lack of significant suppressive effect of dorsal L2-L5 rhizotomy on motor evoked potentials response indicates that descending motor input into alpha-motoneurons is independent on Ia afferent couplings and can independently contribute to increased alpha-motoneuronal excitability. The pharmacology of this effect emphasizes the potent role of GABAergic type B receptors in regulating both the spasticity and rigidity.

The spinal GDNF level is increased after transient spinal cord ischemia in the rat.

Glial cell line-derived neurotrophic factor (GDNF) is known as the most potent neurotrophic factor against injury. We have characterized spinal GDNF changes after ischemia to clarify its possible physiological role against ischemic damage. Spinal ischemia in the rat was produced by cross-clamping of the thoracic aorta together with systemic hypotension. The spinal tissue GDNF level was measured by enzyme-linked immunoabsorbent assay (ELISA) and the localization of GDNF in the tissue was examined by immunohistochemistry. GDNF was increased reaching two peaks after ischemia. The first peak was at 2 hrs after onset of recirculation derived from alpha motor neurons. The second GDNF peak was at 72 hrs provided by astrocytes. These data suggest a necessity of GDNF to increase to protect against ischemic damage, and that activated astrocytes may have an important role in maintaining the GDNF level.

[Propofol anesthesia for an emergent caesarean section in a patient with asthma].

We report a case of emergency caesarean section due to bleeding from placenta praevia under general anesthesia in a patient with asthma. General anesthesia was induced by propofol 150 mg and suxamethonium 80 mg, and operation was started immediately after tracheal intubation under cricoid cartilage pressure. While induction-delivery time took about 270 seconds, Apgar scores at 1 and 5 minutes are 8 and 9 points, respectively. Plasma concentration of propofol in umbilical vein was 0.73 microgram.ml-1 by HPLC. Although anesthetic maintenance was carried out by oxygen-nitrous oxide-isoflurane after delivery, no asthmatic attack was seen throughout the operation. Anesthetic induction by propofol for emergency caesarean section might be safe and useful in a patient with asthma with little effects to neonates.

[A case of total intravenous anesthesia with propofol, fentanyl and ketamine for lateral segmentectomy of the liver under pringle maneuver].

We successfully anesthetized an 80-year-old female for Pringle maneuver which was applied at the time of liver transection and consisted of cross-clamping the hepatoduodenal ligament for 25 minutes and releasing the clamp for 2 minutes until the completion of the liver transection. Anesthesia chosen was total intravenous anesthesia with propofol, fentanyl and ketamine (PFK) in combination with epidural anesthesia. The plasma concentrations of propofol at pre-, intra-, and post-Pringle maneuver under the intravenous infusion of propofol at 3 mg.kg-1.h-1 were 0.84, 1.49, and 1.29 micrograms.ml-1, respectively. All operative procedures were done uneventfully and recovery from anesthesia was prompt after the end of propofol infusion. Transient increases in liver enzymes were seen during early postoperative period, but no signs of hepatic failure were observed. In this patient, PFK anesthesia was useful and safe for the liver transection with Pringle maneuver.

[The inhibition of lidocaine metabolism by various barbiturates in rat hepatic microsome].

To evaluate the effects of various barbiturates on lidocaine metabolism by cytochrome P-450 (P-450), enzyme kinetics were analyzed in an in vitro study using rat hepatic microsomes. Phenobarbital, amobarbital, hexobarbital, pentobarbital, and thiamylal showed the mixed type inhibition of lidocaine metabolism with inhibition constants being 4.89, 1.08, 2.76, 0.77 and 0.65 mM, respectively. Same as lidocaine, all barbiturates used in the present study, corresponding to binding with P-450, induced the I type of spectral change of P-450. Since these did not affect cytochrome C reductase activity, it was suggested that this inhibition of lidocaine metabolism in hepatic microsomes may have been caused by the reduction of activity on P-450 by the barbiturates.

[The plasma concentration measurement of local anesthetics, thiobarbiturates and mexiletine by HPLC with automatic pre-treatment system].

The concentrations of lidocaine, its metabolites (MEGX, GX), mepivacaine, bupivacaine, thiamylal, thiopental and mexiletine in plasma have been determined by HPLC method with automatic pre-treatment system. The drugs carried by pre-treatment mobile phase are trapped by hydrophobic interaction on the pre-treatment column (4 mm i.d. x 30 mm L., tetramethylolmethanetriacrylate resin packing). As sending analytical mobile phase which was conditioned with 0.1 M sodium phosphate buffer (pH 2.1) and acetonitrile by switching the flow changeover valve, the absorped drugs were eluted quickly and introduced to the analytical column (6 mm i.d x 150 mm L., octadecylsilan chemical bond sillica packing). On this column, the drugs were separated and analyzed, then measured by a UV spectrophotmetric detector. The time of switching valve, the wave length of detector for measurement and the buffer/acetonitrile ratio of analytical mobile phase were specific for the drugs, respectively. And high recovery and reproducibility were obtained for using auto-injector, so it has been possible to determine the concentrations of drugs in plasma with absolute calibration curves.

[Examination of the protein binding of lidocaine by gel filtration method].

The protein binding of lidocaine has been studied using gel filtration method. Thermodynamic calculation has been done on results of the experiment. We have found that the primary bond type of lidocaine with serum albumin is hydrophobic interaction, because entropy (delta S) is constantly positive (> 0) under these experimental conditions. The change of the binding ratio between lidocaine and serum albumin with changing pH results from the conformational change of the serum albumin, but the protein binding of lidocaine is also an exothermic reaction. The following conclusions have been obtained. (1) The binding of serum albumin with lidocaine is nonspecific and mainly the result of hydrophobic interaction. (2) At temperature 35 degrees C and pH 7.5, binding constant (K) and binding site (np) of bovine serum albumin with lidocaine are K = 5.9 x 10(3) M-1 and np = 103.5 x 10(-6) M, respectively. (3) Increasing binding ratio with increase in pH is the result of the increase in binding site. This involves the conformational change of albumin, especially that of N-B transition. (4) The binding of serum albumin with lidocaine is an exothermic reaction. Therefore, the binding of lidocaine with serum albumin increases as the temperature decreases.