Rotator cuff tears: what have we learned from animal models?

Rotator cuff tendon tears are among the most common soft tissue injuries that occur at the shoulder. Despite advancements in surgical repair techniques, rotator cuff repairs experience a high rate of failure. The common occurrence of tears and the frequency of re-tears require a further understanding of the mechanisms associated with injuries, healing, and regeneration of the rotator cuff. This paper reviews in vivo studies using the various animal shoulder models of the rat, rabbit, sheep, canine, and primate. These animal models have been used to study intrinsic and extrinsic factors leading to shoulder degeneration, various suture techniques, effects of post-surgical treatment, numerous biologic and synthetic scaffolds, and an assortment of biologic augmentations used to accelerate healing. These effects can be examined in a controlled manner using animal models without other confounding factors that sometimes limit clinical studies. The clinically impactful results will be explained to highlight the specific knowledge gained from using animal models in rotator cuff research.

The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).

The transcription factors of the Rel/NF-kappaB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-kappaB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-kappaB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-kappaB raised the issue of whether NF-kappaB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-kappaB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-x(L). While Bcl-x(L) was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-kappaB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-kappaB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-kappaB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-kappaB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-x(L) and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-kappaB activity.

NADPH-diaphorase-positive neurons in the human inferior colliculus: morphology, distribution and clinical implications.

Using the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction with nitroblue tetrazolium, we provided a detailed investigation of the distribution, dimensional characteristics and morphology of NADPH-d-positive neurons in the three main subdivisions of the human inferior colliculus (IC): central nucleus, pericentral nucleus, and external nucleus. In accordance with their perikaryal diameter, dendritic and axonal morphology, these neurons were categorized as large (averaging up to 45 µm in diameter), medium (20-30 µm), small (13-16 µm) and very small (7-10 µm). Their morphological differences could contribute to varying functionality and processing capacity. Our results support the hypothesis that large and medium NADPH-d-positive cells represent projection neurons, while the small cells correspond to interneurons. Heretofore, the very small NADPH-d-positive neurons have not been described in any species. Their functions-and if they are, indeed, the smallest neurons in the IC of humans-remain to be clarified. Owing to their location, we posit that they are interneurons that connect the large NADPH-d-positive neurons and thereby serve as an anatomical substrate for information exchange and processing before feeding forward to higher brain centers. Our results also suggest that the broad distribution of nitric oxide (NO) synthesis in the human IC is closely tied to the neuromodulatory action of NO on collicular neurotransmitters such as GABA and glutamate, and to calcium-binding proteins such as parvalbumin. A deeper understanding of the relationship between NADPH-d-positive fibers in all IC connections and their co-localization with other neurotransmitters and calcium-binding proteins will assist in better defining the function of NO in the context of its interplay with the cerebral cortex, the sequelae of the aging process and neurodegenerative disorders.

Ultrastructural evidence for destruction in the halo nevus.

Nine halo nevi in various stages of regression were examined by electron microscopy for fine structural evidence of an immunological mechanism of tumor cell destruction and halo formation. Early regressing lesions (Stage I) showed nevus cells associated with infiltrating lymphocytes, monocytes, and plasma cells, but without nevus cell destruction. In later lesions (Stages II and III), vacuolar cytolysis was commonly observed in nevus cells. In Stage III lesions, portions of nevus cells are found within macrophages. The electron microscopic findings of lymphocyte, monocyte, and plasma cell infiltration of the tumor followed by vacuolar cytolysis support the concept of an immune reaction in regressing halo nevi.

Partition of catalase and its peroxidase activities in human red cell membrane: effect of ATP depletion.

Partititon of catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase EC 1.11.1.6) and peroxidase (donor:hydrogen-peroxide oxidoreductase EC 1.11.1.7) activities between the red cell membrane and the cytosol were studied under various experimental conditions. A small but significant amount of catalase (1.6%) was retained on human red cell membranes prepared by hemolysing washed red cells with 30 volumes of 10 mM Tris buffer, pH 7.4. Membrane -bound catalase had a relatively higher peroxidase activity than the soluble enzyme fraction. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the solubilized membranes demonstrated catalase to be a single band with a molecular weight of 60 000. Membranes prepared from adenosine triphosphate-depleted red cells depicted a two to three-fold increase in catalase activity, as well as an increase in 60 000 molecular weight band on polyacrylamide gel electrophoresis. The extra amount of retained catalase was a less efficient peroxidase than found in fresh membranes. The binding of catalase to ATP-depleted red cell membranes was dependent upon both pH and hemolysing ratio. Red cells incubated at pH 7.1 demonstrated a decrease in bound catalase, as did membranes prepared from red cells hemolysed at 1:100 dilution. beta-Mercaptoethanol decreased the catalase activity in the membranes and increased the odianisidine peroxidase activity without any significant effect on the 60 000-dalton band.

Inability of murine melanoma melanosomal "tyrosinase" (L-dopa oxidase) to oxidize tyrosine to melanin in polyacrylamide gel systems.

Melanosomal "tyrosinase" (L-dopa) was isolated from trypsin digest of B-16 mouse melanoma melanosomes, using polyacrylamide gel disc electrophoresis. The enzyme was represented by a single band, having characteristics similar to the T1 dopa-positive band observed when using supernatants of crude melanoma homogenates as the source. When gels with this band were incubated in solutions containing tyrosine and dopa in varying ratios , there was no enhancement of melanin formation by tyrosine when compared with incubations in corresponding concentrations of dopa alone. These data further support previous studies in our laboratory demonstrating an inability of so-called mamalian "tyrosinase" to convert tyrosine to melanin; since this enzyme readily converts L-dopa to melanin, it seems more reasonable to term this enzyme an L-dopa oxidase.

Inability to demonstrate hydroxylation of tyrosine by murine melanoma "tyrosinase" (L-DOPA oxidase), using the tritiated water assay technique.

Validity of the tritiated water assay technique for tyrosine hydroxylase activity as a qualitative method was demonstrated with mushroom tyrosinase. Using this method, isolated murine melanoma "tyrosinase" (L-dopa oxidase) showed no tyrosine hydroxylase activity. This finding supports previous studies in our laboratory which used a variety of histochemical and biochemical methods. The nonenzymatic production of tritiated water caused by tritium exchange with hydrogen peroxide complicates the use of the tritiated water assay technique with crude systems, since hydrogen peroxide is generated by a variety of oxidase reactions. For this reason, previous studies using the tritiated water assay technique with crude systems are ambiguous.

A double-blind placebo-controlled study of 3,4-diaminopyridine in amytrophic lateral sclerosis patients on a rehabilitation unit.

3,4-Diaminopyridine (DAP) enhances acetylcholine release from the nerve terminal and improves conduction in demyelinated axons. In this double-blinded placebo controlled cross over study we examined the effects of DAP combined with inpatient rehabilitation in nine patients with disabling motor weakness due to amyotrophic lateral sclerosis (ALS). A single dose of DAP or placebo was increased daily to the maximum (range: 10-80 mg) tolerated dose; after patients were assessed on the first treatment, the alternate drug was given in the same manner. Functional Independence Measurement (FIM), Ashworth, grip strength, limb strength measurements, nerve conduction studies and speech assessments were initiated 1/2 h after receiving the maximum tolerated dose of DAP or placebo. DAP was tolerated in all patients, but limited by gastrointestinal side effects in four patients. The mean peak serum level was 20.11 (S.D. = 5.11) ng/ml, occurring 1.25 (S.D. = 0.56) h after dose. A statistically significant improvement in FIM and speech assessment scores between admission and discharge occurred. However, no significant differences in clinical or electrophysiologic measures were seen between DAP and placebo treatments. This study suggests that intensive inpatient rehabilitation has a role in the management of patients with ALS, but DAP does not diminish motor impairment.

3,4-diaminopyridine as a treatment for amyotrophic lateral sclerosis.

The slow potassium channel blocker 3,4-diaminopyridine (DAP) enhances acetylcholine release from the nerve terminal and improves conduction in unmyelinated nerve. In this open label pilot study, we examined the effect of DAP combined with inpatient rehabilitation in seven patients with motor weakness due to amyotrophic lateral sclerosis (ALS). A single daily 20 mg oral dose of DAP was gradually increased to the maximum tolerated dose, and serum DAP concentrations were measured. Videotaped motor examination (for subsequent "blinded" review and assignment of a quantitative motor score), Functional Independence Measure (FIM) assessment, nerve conduction studies and neuropsychological evaluations were performed on admission, 1 h after maximum DAP dose, and post-treatment. DAP was tolerated in all patients, though dose was limited by gastrointestinal side effects in five patients. The mean peak serum level was 128 (+/- 50) ng/ml, occurring 1.0 (+/- 0.50) h after dose. A modest but statistically significant (p = 0.045) peak in motor score occurred on DAP. A significant (p = 0.045) improvement from baseline in FIM performance was apparent with DAP. Nerve conduction studies showed small increases in evoked response amplitudes and conduction velocities on DAP, but they did not reach statistical significance. No cognitive or affective changes were apparent. This unblinded pilot study shows that DAP is tolerated in ALS patients, and may be associated with functional and electrophysiologic improvement.

Robot-aided neurorehabilitation: from evidence-based to science-based rehabilitation.

There is no "magic bullet" in rehabilitation. In the absence of direct neural transplants, neurological rehabilitation is an arduous process. We have pioneered the clinical application of robotics in stroke rehabilitation and have shown evidence of the positive impact of targeted exercise on stroke recovery. In this article, we will review results obtained in the initial clinical trials with 96 stroke patients at the Burke Rehabilitation Hospital. We will provide evidence that robot-aided training enhances recovery, that this enhanced recovery is sustained in the long term, and that this recovery is not due to a general physiological improvement--in fact, it appears to be limb and muscle group specific. An evidence-based approach must now segue into a more scientific approach to stroke rehabilitation. Given the length of the required protocols and patients' variability and limited census, the practical limitations of the evidence-based approach are self-evident and extend trials for years. Each patient and lesion is unique in stroke rehabilitation, so there is no reason to believe that a "one-size-fits-all" optimal treatment exists. To optimize therapy for individual patients, we need science-based models. In this article, we will summarize the scientific tools and models that we are investigating and present some of the results to date.

Overview of clinical trials with MIT-MANUS: a robot-aided neuro-rehabilitation facility.

We are applying robotics and information technology to assist, enhance, and quantify neuro-rehabilitation. Our goal is a new class of interactive, user-affectionate clinical devices designed not only for evaluating patients, but also-for delivering meaningful therapy via engaging "video games". Notably, the novel robot MIT-MANUS has been designed and programmed for clinical neurological applications, and has undergone extensive clinical trials for more than four years at Burke Rehabilitation Hospital - White Plains, NY. This paper will review results of the first clinical trial of 20 patients, which showed that: - Stroke patients treated daily with additional robot-aided therapy during acute rehabilitation had improved outcome in motor activity at hospital discharge, when compared to a control group that received only standard acute rehabilitation treatment. - This improved outcome was sustained after three years. - The neuro-recovery process continued far beyond the commonly accepted 3 months post-stroke interval.

Parvalbumin-immunoreactive neurons in the human claustrum.

The morphology and distribution of parvalbumin-immunoreactive neurons (PV-ir) were studied in the human claustrum. PV-ir neurons were observed throughout the claustrum, with the highest numbers noted in the central (broadest) portion as compared with the dorsal and ventral aspects. Reaction product was evident in the neuronal perikarya, dendritic processes, and spines. In the majority of these labeled neurons, the cytoplasm was devoid of lipofuscin pigment. Cell bodies varied widely in both shape and size, ranging from oval and small, to multipolar and large. PV-ir neurons were classified into two groups, primarily based on dendritic morphology: spiny neurons with long and straight dendrites, and aspiny neurons with thin and curving dendritic processes. PV-ir fibers were seen throughout the neuropil, with many immuno-positive puncta noted.

Neuropeptide Y immunoreactivity in the cat claustrum: A light- and electron-microscopic investigation.

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular, primary sensory areas. However, despite renewed and growing interest amongst investigators, there remains a paucity of data concerning its peptidergic profile. The aim of the present study was to examine the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir) neurons and fibers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All animals received human and ethical treatment in accordance with the Principles of Laboratory Animal Care. Subjects were irreversibly anesthetized and transcardially perfused with fixative solution containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postfixed and sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin-biotin-peroxidase complex method adopted by our Department of Anatomy, Histology and Embryology. NPY-ir neurons and fibers were found to be diffusely distributed throughout the claustrum, with no obvious topographic or functional patterning other than larger numbers in its central/broadest part (stereotaxic planes A12-A16). Neurons were generally classified by diameter into three sizes: small (under 17 µm), medium (17-25 µm) and large (over 25 µm). Staining density is varied with some neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that gender-specific differences were not observed.

MicroRNAs in platelet production and activation.

Recent work by the Encyclopedia of DNA Elements project showed that non-protein-coding RNAs account for an unexpectedly large proportion of the human genome. Among these non-coding RNAs are microRNAs (miRNAs), which are small RNA molecules that modulate protein expression by degrading mRNA or repressing mRNA translation. MiRNAs have been shown to play important roles in hematopoiesis including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. Additionally, disordered miRNA biogenesis and quantitative or qualitative alterations in miRNAs and their targets are associated with hematological pathologies. Platelets contain machinery to process pre-miRNAs into mature miRNAs, and specific platelet miRNA levels have been found to correlate with platelet reactivity. This review summarizes the current state of knowledge of miRNAs in megakaryocytes and platelets, and the exciting possibilities for future megakaryocyte-platelet transcriptome research.