Angiotensin II type 1 receptor localizes at the blood-bile barrier in humans and pigs.

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin-angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood-bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability.

Insertional anatomy of the anterior medial collateral ligament on the sublime tubercle of the elbow.

BACKGROUND: Acute injuries to the anterior medial collateral ligament (AMCL) can occur due to valgus trauma or during other dislocating events to the elbow. AMCL lesions are often associated with bony lesions, such as radial head fractures or fractures of the coronoid process. We analyzed the insertion of the AMCL on the sublime tubercle in relation to surrounding osseous structures. We aimed to increase the understanding of the involvement of the AMCL in bony lesions to the sublime tubercle. METHODS: We investigated 86 elbows from 43 embalmed human specimens. We measured the most ventral extensions of the AMCL at the sublime tubercle in relation to a clearly defined and reproducible landmark. We used as our landmark a horizontal line (baseline) originating on the lesser sigmoid notch in a right angle to the ulnar ridge. RESULTS: The mean distance of the coronoid process tip to the baseline was 4.0 mm (standard deviation [SD], 1.3 mm; range, 1.4-6.7 mm). The mean distance of the ventral extension of the AMCL to the horizontal line was 3.7 mm (SD, 2.6 mm; range: 9.4-2.2 mm). The mean horizontal distance between the ventral aspect of the AMCL and the coronoid tip was 13.7 mm (SD, 2.5 mm; range, 7.7-20.5 mm). CONCLUSIONS: We present a detailed description of the insertional anatomy of the AMCL at the sublime tubercle. These values could be helpful for classifications of coronoid fractures and to estimate the involvement of the AMCL in fractures of the sublime tubercle.

The radial head size in relation to osseous landmarks of the forearm.

PURPOSE: Radial head fractures are regularly treated with radial head arthroplasty. To prevent limited motion or pain, the implant's size should match its normal anatomy. Preoperative estimation of the radial head size helps in finding the correct head component. The aim of this study was to measure bony landmarks in proximity to the radial head to estimate the required size of a prosthesis preoperatively. METHODS: Anatomical landmarks on 82 elbows from 41 embalmed specimens (19 male, 22 female) were measured using a digital caliper after removal of the specimens' tissue: the largest and smallest radial head diameter, length of the radius (styloid tip to radial head articular surface), and the length of the ulna (styloid tip to coronoid base). Additionally, cranio-caudal and antero-posterior diameters of the capitulum on scaled lateral elbow X-ray images were measured. RESULTS: The mean largest and smallest radial head diameters were 24.2 mm (± 2.2, range 19.9-30.3; ICC = 0.992) and 22.5 mm (± 2.0, range 18.9-27.5; ICC = 0.985). The mean radius length was 23.8 cm (± 1.6, range 20.1-27.1; ICC = 0.986), and the mean ulna length was 23.1 cm (± 1.6, range 19.3-26.3; ICC = 0.969). The mean antero-posterior capitulum diameter was 16.2 mm (± 2.4, range 10.4-21.0; ICC = 0.506), and the mean cranio-caudal diameter was 17.0 mm (± 3.3, range 10.0-23.9; ICC = 0.529). The highest correlation to radial head diameters could be shown for diameters of the contralateral radial head and the radius length. CONCLUSIONS: For preoperative estimation of the radial head, the diameters of the contralateral radial head or the radius length are the most accurate.

Cav2.3 (R-Type) Calcium Channels are Critical for Mediating Anticonvulsive and Neuroprotective Properties of Lamotrigine In Vivo.

BACKGROUND/AIMS: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED), however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Cav2.3 (R-type) calcium currents, which contribute to kainic-acid- (KA) induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to topiramate and lacosamide in Cav2.3-deficient mice and controls on KA-induced seizures. METHODS: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg [and 30 mg/kg] KA. One hour before KA injection, mice were pretreated with either 30 mg/kg LTG, 50 mg/kg topiramate (TPM) or 30 mg/kg lacosamide (LSM). RESULTS: Ablation of Cav2.3 reduced total seizure scores by 28.6% (p=0.0012) and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p=0.02). In Cav2.3-deficient mice LTG pretreatment increased seizure activity by 22.1% (p=0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p=0.02). All three tested AEDs reduced seizure activity in control mice, however only the non-calcium channel modulating AED, LSM had an anticonvulsive effect in Cav2.3-deficient mice. Furthermore LTG altered electrocorticographic parameters differently in the two genotypes, decreasing relative power of ictal spikes in control mice compared to Cav2.3-defcient mice. CONCLUSION: These findings give first in vivo evidence for an essential role for Cav2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Cav2.3-deficient mice resulting in increased neurotoxicity in the CA1 region. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation, observed in clinical practice.

Intact blood-brain barrier during spontaneous attacks of migraine without aura: a 3T DCE-MRI study.

BACKGROUND AND PURPOSE: The integrity of the blood-brain barrier (BBB) has been questioned in migraine, but BBB permeability has never been investigated during spontaneous migraine attacks. In the present study, BBB permeability during spontaneous attacks of migraine without aura was investigated compared to an interictal state. METHODS: Seventy-four patients suffering from migraine without aura were recruited to participate in this cross-sectional dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) study. The patients were instructed to report at the hospital for DCE-MRI scan during and outside of a spontaneous migraine attack. The primary end-point was a difference in the BBB permeability (ml/100 g/min) between the attack and the headache-free days. The permeability was assessed in five different regions of interest (ROIs) located in the anterior, middle and posterior cerebral area, brain stem, posterior pons and whole brain. The paired samples t test was used to compare Ki (permeability) values between the attack and headache-free days. RESULTS: Nineteen patients completed the study. Median time from onset of migraine attack to scan was 6.5 h (range 4.0-15.5 h). No change in the mean BBB permeability (ml/100 g/min) was found between the attack and the headache-free days in any of the measured ROIs. No relationship between the pain side or intensity and BBB permeability was found in 15 patients with unilateral pain during the examined attack. CONCLUSIONS: It was demonstrated that the BBB permeability during spontaneous migraine attacks without aura was unchanged.

Radial shortening osteotomy reduces radiocapitellar contact pressures while preserving valgus stability of the elbow.

PURPOSE: Shortening osteotomy of the proximal radius might represent a potential salvage procedure in symptomatic radiocapitellar osteoarthritis, which could decrease radiocapitellar load while preserving the native radial head. In an in-vitro biomechanical investigation, we sought to determine whether shortening osteotomy of the proximal radius (1) decreases the radiocapitellar joint pressure upon axial loading and (2) retains valgus stability of the elbow. In addition, the anatomic configuration of the lesser sigmoid notch was evaluated to assess possible contraindications. METHODS: Axial loading (0-400 N) and valgus torque (7.5 N m) over the full range of motion were applied to 14 fresh-frozen specimens before and after shortening osteotomy of the proximal radius by 2.5 mm. Radiocapitellar and ulnohumeral load distribution during axial compression was evaluated using a digital pressure mapping sensor. Valgus displacement was analyzed with a 3D camera system. The inclination angle (α) of the lesser sigmoid notch was assessed via 50 CT scans. RESULTS: Up to axial loading of 250 N, shortening osteotomy caused a significant decrease in radiocapitellar contact pressures (p < 0.041). Valgus stability of specimens did not differ before and after shortening osteotomy (n.s.). The mean inclination angle (α) of the lesser sigmoid notch was 11.3° ± 6.3°. 46% had an inclination angle of ≤ 10° (type I). 46% had an inclination angle of 11°-20° (type II). In 8%, the inclination angle was >20° (type III). CONCLUSION: Shortening osteotomy of the proximal radius can decrease radiocapitellar contact pressures during axial loading of up to 250 N. Primary valgus stability is not relevantly influenced by this procedure. In few patients, shortening osteotomy may cause radioulnar impingement of the radial head at the distal edge of the lesser sigmoid notch due to an inclination angle of >20°. Shortening osteotomy might be a promising treatment option to decrease pain levels in case of isolated radiocapitellar osteoarthritis.

Biomechanical comparison of transosseous re-fixation of the deep fibres of the distal radioulnar ligaments versus deep and superficial fibres: a cadaver study.

PURPOSE: We hypothesized that the re-fixation of the deep and superficial fibres of the distal radioulnar ligaments provide improved stability compared to reconstruction of the deep fibres alone. METHODS: Fourteen fresh-frozen cadaver upper extremities were used for biomechanical testing. Transosseous re-fixation of the deep fibres of the distal radioulnar ligaments alone (single mattress suture group; n = 7) was compared to the transosseous re-attachment of the deep and superficial fibres (double mattress suture group; n = 7). Cyclic load application provoked palmar translation of the radius with respect to the rigidly affixed ulna. Creep, stiffness, and hysteresis were obtained from the load-deformation curves, respectively. Testing was done in neutral forearm rotation, 60° pronation, and 60° supination. RESULTS: The re-fixation techniques did not differ significantly regarding the viscoelastic parameters creep, hysteresis, and stiffness. Several significant differences of one cycle to the consecutive one within each re-fixation group could be detected especially for creep and hysteresis. No significant differences between the different forearm positions could be detected for each viscoelastic parameter. CONCLUSIONS: The re-fixation techniques did not differ significantly regarding creep, hysteresis, and stiffness. This means that the additional re-attachment of the superficial fibres may not provide greater stability to the DRUJ. Bearing in mind that the study was a cadaver examination with a limited number of specimens we may suppose that the re-attachment of the superficial fibres seem to be unnecessary. A gradual decline of creep and hysteresis from first to last loading-unloading cycle is to be expected and typical of ligaments which are viscoelastic.

Paclitaxel inhibits mRNA transport in axons.

Paclitaxel is an integral component of solid tumor treatment. This chemotherapeutic agent provokes an often irreversible peripheral sensory neuropathy with pathological features of distal axonal degeneration. Current pathological concepts assume that polymerization of axonal microtubules and mitochondrial dysfunction contributes to the development of paclitaxel-induced peripheral neuropathy. The relationship, however, between microtubule stabilization, mitotoxicity and axonal degeneration is still not completely understood. To explore the function of axonal mitochondria we treated transgenic mice that harbor cyan fluorescent protein (CFP)-labeled neuronal mitochondria with repeated doses of paclitaxel and assessed neuropathic changes by nerve conduction and histological studies. In addition, mitochondrial content and morphology was determined by ex vivo imaging of axons containing CFP-labeled mitochondria. Using quantitative RT-PCR and fluorescence-labeled mRNA we determined axonal mRNA transport of nuclear encoded mitochondrial proteins. Prolonged treatment with high doses of paclitaxel-induced a predominant sensory neuropathy in mice. Although mitochondrial velocity in axons per se was not altered, we observed significant changes in mitochondrial morphology, suggesting that paclitaxel treatment impairs the dynamics of axonal mitochondria. These changes were caused by decreased levels of nuclear encoded mRNA, including the mitochondrial fusion/fission machinery. Moreover, impaired axonal mRNA transport in vitro resulted in mitochondrial dysfunction and subsequent axonal degeneration. Taken together, our experiments provide evidence that disrupted axonal transport of nuclear derived mRNA plays a crucial role in the pathogenesis of paclitaxel-induced sensory neuropathy.

The course of the median and radial nerve across the elbow: an anatomic study.

INTRODUCTION: Nerve transection has been described as complication of arthroscopic elbow arthrolysis. Therefore, the goal of this study was to define bony landmarks for intraoperative orientation regarding the location of the median and radial nerve. METHODS: In 22 formalin-fixated upper extremities, the radial and median nerves were dissected and marked with respect to their native course. A 3D X-ray scan was performed. The distances of the radial nerve to the radial head (R1), the capitulum (R2), and its lateral border (RC) were measured. The location of the radial nerve in relation to the transversal diameter of the humeral condyle (HC) was calculated. Similarly, the distances of the median nerve to the trochlea (M1), the medial border of the trochlea (M2), and its relation to HC were calculated. RESULTS: The mean value for R1 was 8 mm (±2.9 mm), for R2 was 11.3 mm (±3.8 mm), and for RC was 10.6 mm (±5.1 mm). RC/HC averaged 24 % (±11 %). M1 averaged 11.7 mm (±5.2 mm), and M2 was 2.4 mm (±4.1 mm). M2/HC averaged 6 % (±9 %). CONCLUSIONS: The radial nerve is located ventral to the central third of the capitulum. The median nerve lies ventral to the medial quarter of the humeral condyle. When performing arthroscopic arthrolysis, this information should be kept in mind during anterior capsulectomy.

Do the radial head prosthesis components fit with the anatomical structures of the proximal radioulnar joint?

PURPOSE: The fitting accuracy of radial head components has been investigated in the capitulo-radial joint, and reduced contact after prosthetic replacement of the radial head has been observed. The kinematics of the proximal radioulnar joint (PRUJ) are affected by radial head arthroplasty as well, but have not yet been investigated in this regard. METHODS: The elbow joints of 60 upper extremities of formalin-fixed body donors were disarticulated to obtain a good view of the PRUJ. Each specimen was mounted on the examining table and radial head position in the native PRUJ was assessed in neutral position, full pronation, and full supination. Measurements were repeated after implantation of mono- and bi-polar prostheses. RESULTS: Analysis of the distribution of the joint contacts in the compartments showed significant differences after radial head replacement. In comparison to the native joint, after bipolar and monopolar radial head replacement, the physiological shift of the proximal radius was altered. DISCUSSION: The physiological shift of the joint contact of the radial head from anterior to posterior during forearm rotation that was found in the native joint in our cadaver model was not observed after prosthetic replacement. With higher conformity and physiological kinematic of radial head prostheses, possibly lower shear forces and lower contact pressures would be generated. CLINICAL RELEVANCE: The tested radial head prostheses do not replicate the physiological kinematics of the radial head. Further development in the prosthesis design has to be made. The meticulous reconstruction of the annular ligament seems to be of importance to increase joint contact.

Neonatal mouse ileum: functional properties and protein composition of the contractile machinery.

BACKGROUND: Immature motility of the ileum may contribute to life-threatening diseases. Little is known about the normal biomechanics of the neonatal ileum in relation to the protein composition of its contractile machinery. METHODS: We analyzed the tissue architecture, the biomechanics in intact and ß-escin-permeabilized preparations, and the protein composition in neonatal (P0) and adult murine ileum. RESULTS: Muscle thickness of the P0 ileum was -50% of the adult ileum and passive compliance was higher. Carbachol- and KCl-elicited contractions were tonic rather than phasic as in the adult. Ca(2+) sensitivity was higher and relaxation rate was slower in ß-escin-permeabilized P0 compared with adult ileum. The expression level of ß-actin relative to α-actin was higher, and those of total actin, myosin, myosin light chain kinase, the catalytic subunit of myosin phosphatase and telokin were lower compared with the adult. The expression level of MYPT1 was similar, but P0 ileum expressed only the M133; the adult ileum also expressed the M130 isoform. CONCLUSION: The mechanical features and protein composition of the P0 ileum are similar to those of adult tonic smooth muscles. We propose that this is highly adaptive during fetal life allowing the small intestine to act predominantly as a container.

Course of the radial nerve in relation to the center of rotation of the elbow--the need for a rational safe zone for lateral pin placement.

PURPOSE: To investigate the course and variability of the radial nerve along the lateral humerus in relation to the center of rotation of the elbow joint in the context of lateral pin placement for hinged external fixation. METHODS: A total of 95 formalin-fixed upper extremities were dissected. The course of the radial nerve along the lateral aspect of the humerus was measured at 3 landmarks with respect to the center of rotation of the elbow. We analyzed the data and the landmark positions correlated with the length of the humerus. RESULTS: The measured positions of 3 landmarks of the radial nerve in the lateral aspect of the humerus ranged from 19% to 43% of the length of the humerus and were located, on average, 6.0, 9.7, and 13.5 cm from the lateral center of rotation. CONCLUSIONS: These data help predict the humeral course of the radial nerve and define a safe zone for pin implantation. However, because of variability in the course of the radial nerve, a safe zone cannot fully ensure prevention of iatrogenic injury to the nerve. The safest method of pin application remains mini-open dissection and visual implantation. CLINICAL RELEVANCE: Based on this cadaveric study, it is not possible to define a rational safe zone. The safest method of pin application for dynamic external fixation of the elbow is to perform a mini-open dissection with direct visualization.

Cav 2.3 (R-type) calcium channels are critical for mediating anticonvulsive and neuroprotective properties of lamotrigine in vivo.

PURPOSE: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED); however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Cav 2.3 (R-type) calcium currents, which contribute to kainic-acid (KA)-induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to two other AEDs in Cav 2.3-deficient mice and controls on KA-induced seizures. METHODS: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg (and 30 mg/kg) KA. One hour before KA injection, mice were pretreated with 30 mg/kg LTG, 50 mg/kg topiramate (TPM), or 30 mg/kg lacosamide (LSM). KEY FINDINGS: Ablation of Cav 2.3 reduced total seizure scores by 28.6% (p = 0.0012), and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p = 0.02). In Cav 2.3-deficient mice, LTG pretreatment increased seizure activity by 22.1% (p = 0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p = 0.02). All three AEDs reduced seizure activity in control mice; however, only the non-calcium channel modulating AED, LSM, had an anticonvulsive effect in Cav 2.3-deficient mice. Furthermore, LTG altered electrocorticographic parameters differently in the two genotypes: decreasing relative power of ictal spikes in control mice but increasing relative power of high frequency fast ripple discharges during seizures in Cav 2.3-deficient mice. SIGNIFICANCE: These findings provided the first in vivo evidence for an essential role for Cav 2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Cav 2.3-deficient mice by increasing high frequency components of seizures, resulting in increased neurotoxicity in the CA1. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation observed in clinical practice.

C3 exoenzyme lacks effects on peripheral axon regeneration in vivo.

Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP-ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3bot suggesting an inflammatory component in the inhibition of axon growth. C3bot or C3E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3E174Q . Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth-promoting environment.

A new paradigm of hearing loss and preservation with cochlear implants: Learnings from fundamental studies and clinical research.

In 2010 Cochlear initiated a coordinated preclinical research program to identify the factors and underlying mechanisms of acoustic hearing loss following cochlear implantation and device use. At its inception the program was structured around several major hypotheses implicated in the loss of acoustic hearing. The understanding of causes evolved over the course of the program, leading to an increased appreciation of the role of the biological response in post-implant hearing loss. A systematic approach was developed which mapped the cochlear implant journey along a timeline that considers all events in an individual's hearing history. By evaluating the available data in this context, rather than by discrete hypothesis testing, causative and associated factors may be more readily detected. This approach presents opportunities for more effective research management and may aid in identifying new prospects for intervention. Many of the outcomes of the research program apply beyond preservation of acoustic hearing to factors important to overall cochlear health and considerations for future therapies.

Morphological, ultrastructural, genetic characteristics and remarkably low prevalence of macroscopic Sarcocystis species isolated from sheep and goats in Kurdistan region, Iraq.

Introduction: Sarcocystis is a genus of cyst-forming parasites that infest both humans and livestock. Some parasites cause clinical and subclinical diseases in their hosts, resulting in economic losses. Methods: Esophagus, diaphragm, and skeletal muscle from slaughtered sheep and goats were examined macroscopically, microscopically, and ultrastructurally and subjected to DNA analysis. Results: We isolated macrocysts of S. gigantea and of S. caprafelis moulei from naturally infected sheep (Ovis aries) and goats (Capra hircus). The macrocyst wall thickness was 18.9 µm in sheep and 15.3 µm in goats, and consisted of an inner Periodic acid Schiff- (PAS) negative primary wall and an outer glycoconjugates containing i.e. PAS-positive secondary wall. The walls inner surface was compartmentalized and filled with bradyzoites. In S. gigantea the bradyzoites were approximently 12.3 x 2.6 µm in size, while in S. caprafelis moulei they were 13.9 x 4.4 µm. Ultrastructurally, both species have nearly identical morphology: cauliflower-like protrusions with numerous microtubules and often dendritic-like filaments, branching from the primary wall. The 18S rRNA gene in S. gigantea was 85.9% identical to that in S. medusiformis and 80.4% to the S. caprafelis moulei gene. The 28S rRNA gene in S. gigantea was 94.6% identical to that in S. medusiformis and 97.3% to the S. caprafelis moulei. Conclusion: This study is the first to (i) detail the ultrastructure of the macrocyst wall of S. caprafelis moulei, (ii) identify S. medusiformis in Iraqi sheep, and (iii) compare the prevalence of macroscopic Sarcocystis at different time periods within the same region. A positive finding was the reduction of macroscopic sarcocystosis occurrences (0.01% in sheep and 0.02% in goats) compared to our previous data from 1992 (4.1%: sheep, 33.6%: goats).

Treadmill training of rats after sciatic nerve graft does not alter accuracy of muscle reinnervation.

Background and purpose: After peripheral nerve lesions, surgical reconstruction facilitates axonal regeneration and motor reinnervation. However, functional recovery is impaired by aberrant reinnervation. Materials and methods: We tested whether training therapy by treadmill exercise (9 × 250 m/week) before (run-idle), after (idle-run), or both before and after (run-run) sciatic nerve graft improves the accuracy of reinnervation in rats. Female Lewis rats (LEW/SsNHsd) were either trained for 12 weeks (run) or not trained (kept under control conditions, idle). The right sciatic nerves were then excised and reconstructed with 5 mm of a congenic allograft. One week later, training started in the run-run and idle-run groups for another 12 weeks. No further training was conducted in the run-idle and idle-idle groups. Reinnervation was measured using the following parameters: counting of retrogradely labeled motoneurons, walking track analysis, and compound muscle action potential (CMAP) recordings. Results: In intact rats, the common fibular (peroneal) and the soleus nerve received axons from 549 ± 83 motoneurons. In the run-idle group, 94% of these motoneurons had regenerated 13 weeks after the nerve graft. In the idle-run group, 81% of the normal number of motoneurons had regenerated into the denervated musculature and 87% in both run-run and idle-idle groups. Despite reinnervation, functional outcome was poor: walking tracks indicated no functional improvement of motion in any group. However, in the operated hindlimb of run-idle rats, the CMAP of the soleus muscle reached 11.9 mV (normal 16.3 mV), yet only 6.3-8.1 mV in the other groups. Conclusion: Treadmill training neither altered the accuracy of reinnervation nor the functional recovery, and pre-operative training (run-idle) led to a higher motor unit activation after regeneration.

Treatment With Nimodipine or FK506 After Facial Nerve Repair Neither Improves Accuracy of Reinnervation Nor Recovery of Mimetic Function in Rats.

Purpose: Nimodipine and FK506 (Tacrolimus) are drugs that have been reported to accelerate peripheral nerve regeneration. We therefore tested these substances aiming to improve the final functional outcome of motoric reinnervation after facial nerve injury. Methods: In 18 female rats, the transected facial nerve was repaired by an artificial nerve conduit. The rats were then treated with either placebo, nimodipine, or FK506, for 56 days. Facial motoneurons were pre-operatively double-labeled by Fluoro-Gold and again 56 days post-operation by Fast-Blue to measure the cytological accuracy of reinnervation. The whisking motion of the vibrissae was analyzed to assess the quality of functional recovery. Results: On the non-operated side, 93-97% of those facial nerve motoneurons innervating the vibrissae were double-labeled. On the operated side, double-labeling only amounted to 38% (placebo), 40% (nimodipine), and 39% (FK506), indicating severe misdirection of reinnervation. Regardless of post-operative drug or placebo therapy, the whisking frequency reached 83-100% of the normal value (6.0 Hz), but whisking amplitude was reduced to 33-48% while whisking velocity reached 39-66% of the normal values. Compared to placebo, statistically neither nimodipine nor FK506 improved accuracy of reinnervation and function recovery. Conclusion: Despite previous, positive data on the speed and quantity of axonal regeneration, nimodipine and FK506 do not improve the final functional outcome of motoric reinnervation in rats.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.