Penile neurovascular structure revisited: immunohistochemical studies with three-dimensional reconstruction.

Penile erection is a neurovascular phenomenon that requires well coordinated and functional interaction between penile vascular and nervous systems. In order to provide a useful tool to examine pathologic changes in the erectile tissue, mainly focusing on penile neurovascular dysfunction, we established the technique to determine the differential distribution of endothelial cells, smooth muscle cells, pericytes, and nerve fibers in the mouse penis using immunohistochemical staining with three-dimensional reconstruction. Immunofluorescent staining of penile tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α -actin (SMA, a smooth muscle cell marker), NG2 (a pericyte marker), or ßIII-tubulin (a neuronal marker). We reconstructed three-dimensional images of penile vascular or neurovascular system from stacks of two-dimensional images, which allows volume rendering and provides reliable anatomic information. CD31-positive endothelial cells, SMA-positive smooth muscle cells, and NG2-positive pericytes were evenly distributed and composed sinusoidal or venous wall. However, the endothelial layer of the cavernous artery or dorsal artery was mainly covered with smooth muscle cells and rarely associated with pericytes. The reconstructed three-dimensional images clearly visualized typical wavy appearance of nerve fibers that evenly innervate to cavernous sinusoids, cavernous artery, dorsal vein, and dorsal artery. We observed a significant decrease in CD31-positive endothelial cells, NG2-positive pericytes, and ßIII-tubulin-positive nerve fibers in the penis of diabetic mice compared with those in normal condition. Our protocol for immunofluorescent staining with three-dimensional reconstruction will allow a better understanding of the penile neurovascular anatomy and may constitute a standard technique to determine the efficacy of candidate therapeutics targeting therapeutic angiogenesis or neural regeneration.

Calorie restriction reverses age-related alteration of cavernous neurovascular structure in the rat.

Calorie restriction (CR) refers to a reduction of calorie intake without compromising essential nutrients to avoid malnutrition. CR has been established as a non-genetic method of altering longevity and attenuating biological changes associated with aging. Aging is also an important risk factor for erectile dysfunction. The aim of this study was to examine whether CR diet can reverse the age-related alterations of erectile tissue in the aged rat. Four groups of rats were used: young rats (7 months) + ad libitum, aged rats (22 months) + ad libitum, young rats + CR diet, and aged rats + CR diet. The ad libitum group had free access to both food and water, and CR groups were fed 60% of the food intake of their ad libitum littermates, starting from 6 weeks before sacrifice. The penis was harvested and stained with antibodies to von Willebrand factor, smooth muscle α-actin, platelet-derived growth factor receptor-ß, phospho-eNOS, nNOS, and neurofilament. We also performed Masson trichrome staining and TUNEL assay. The blood samples were collected for the measurement of serum total testosterone level. The contents of endothelial cells, smooth muscle cells, pericytes, and neuronal cells as well as serum testosterone levels were significantly lower in the penis of aged rats than in their young littermates. CR significantly restored cavernous endothelial cells, smooth muscle cells, pericytes, and neuronal cell contents and decreased cavernous endothelial cell apoptosis and fibrosis in both young and aged rats. CR also increased serum testosterone level in aged rats, but not in young rats. CR successfully improved age-related derangements in penile neurovascular structures and hormonal disturbance. Along with a variety of lifestyle modifications, our study gave us a scientific rationale for CR as a non-pharmaceutical strategy to reprogram damaged erectile tissue toward neurovascular repair in aged men.

Establishment of in vitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy.

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes-induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an in vitro model of ED for the study of high-glucose-induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal-glucose (5 mmoL) or high-glucose (30 mmoL) condition for 48 h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high-glucose condition. To study cellular interaction between MCECs and MCPs, co-culture systems including indirect contact, indirect non-contact, and direct mixed co-culture system, were established. We observed impaired tube formation and increased permeability in MCECs-MCPs co-culture exposed to high-glucose condition. To evaluate the effect of high-glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS-positive nerve fibers were significantly lower in MPG tissues exposed to the high-glucose condition than in the tissues exposed to the normal-glucose condition. We believe that in vitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

Curcumin exerts antidifferentiation effect through AMPKalpha-PPAR-gamma in 3T3-L1 adipocytes and antiproliferatory effect through AMPKalpha-COX-2 in cancer cells.

Curcumin has been reported to have the potential to prevent obesity as well as cancers. The downstream targets regulated by AMP-activated protein kinase (AMPK) for inhibiting adipocyte differentiation or cancer cell proliferation of curcumin were investigated. The activation of AMPK by curcumin was crucial for the inhibition of differentiation or growth in both adipocytes and cancer cells. Stimulation of AMPK by curcumin resulted in the down-regulation of PPAR (peroxisome proliferator-activated receptor)-gamma in 3T3-L1 adipocytes and the decrease in COX-2 in MCF-7 cells. Application of a synthetic AMPK activator also supported the evidence that AMPK acts as an upstream signal of PPAR-gamma in 3T3-L1 adipocytes. In cancer cells, AMPK was found to act as a regulator of ERK1/2, p38, and COX-2. Regulation of AMPK and its downstream targets such as PPAR-gamma, Mapkinases, and COX-2 by curcumin appears to be important in controlling adipocytes and cancerous cells.

Calf-contouring surgery of gastrocnemius hypertrophy: selective neurectomy of the sural nerve.

Generally, Asians tend to have obese calves that are shorter and thicker than those of Caucasians. The cause of the enlarged calves is either an excess of subcutaneous fat or calf muscular hypertrophy, but some patients have both conditions. These features are accentuated by the contraction of the calf muscles when patients stand in tiptoe position or wear high heels. In the case of calf muscular hypertrophy without excessive subcutaneous fat, manipulation of the calf muscle is an effective method for reducing calf circumference. From January 2005 to December 2006, the authors performed selective sural neurectomy for 20 patients who complained of obese calves. Using a popliteal incision, the sural nerve branches to the medial and/or lateral gastrocnemius muscles were dissected from the posterior tibial nerve. Using a nerve stimulator, the branches with the most contractile portions were resected in 1 cm lengths at the distal ends. One medial branch was resected in 15 patients, and two branches were resected in 5 patients. In the case of a lateral neurectomy, only one branch was resected. Ultrasound-assisted liposuction was combined for five patients who also showed excessive subcutaneous fat. The patients ranged in age from 19 to 29 years (mean, 23 years). The follow-up period varied from 6 to 18 months. The circumferential change was checked 6 months postoperatively 15 cm below the medial condyle and 15 cm above the medial malleolus. These average circumferences were reduced, respectively, from 36.6 and 32.5 cm to 35.5 and 32.2 cm. In gait analysis performed at 6 months postoperatively, the calf muscle power was slightly reduced to 95% of the preoperative state, but still remained above the normal range. Subjectively, no patient complained of muscle weakness or gait disturbance. One patient showed lower leg edema after mountain climbing that subsided after 1 day of rest. The authors believe a selective neurectomy technique can be an effective method for treating obese calves.

Effect of resistant starch from corn or rice on glucose control, colonic events, and blood lipid concentrations in streptozotocin-induced diabetic rats.

To examine the effect of two types of resistant starch on blood glucose and insulin levels, colonic events, hypolipidemic actions and humoral immune responses, Sprague-Dawley streptozotocin-induced diabetic rats were fed diet containing resistant starch from corn or rice. The marked body weight loss by inducing diabetes was not recovered by feeding resistant starch, even though there are no differences in food intakes compared to the non-diabetic control rats. No significant effect of resistant starch feeding on blood glucose and insulin was found. Even though the length of small intestines, and cecum, colon and rectum together with the tissue weight of cecum were not affected by feeding resistant starch, the intestinal transit time was markedly shortened by both types of resistant starch and resistant starch from corn had a more pronounced effect. The short chain fatty acids in the intestinal contents did not appear to be different among the groups. Nonetheless, both of resistant starch from corn and rice significantly lowered plasma total lipid and cholesterol concentrations compared to the diabetic control. The total liver cholesterol lowering effect was observed with resistant starch from rice. Neither immunoglobulin G nor C(3) were influenced by resistant starch.

Effects of cysteine on amino acid concentrations and transsulfuration enzyme activities in rat liver with protein-calorie malnutrition.

The changes in amino acid concentrations and transsulfuration enzyme activities in liver were investigated after 4-week fed on 23% casein diet (control group) and 5% casein diet without (protein-calorie malnutrition, PCM group) or with (PCMC group) oral administration of cysteine, 250 mg/kg (twice daily, starting from the fourth week) using rats as an animal model. By supplementation with cysteine in PCM rats (PCMC group), cysteine level was elevated almost close to the control level, and glutathione (GSH), aspartic acid and serine levels were restored greater than the control levels. The measurement of transsulfuration enzyme activities exhibited that gamma-glutamylcysteine ligase (gamma-GCL) activity was up-regulated in rats with protein restriction (PCM group), and cysteine supplementation (PCMC group) down-regulated to the control level. One-week supplementation of cysteine (PCMC group) significantly down-regulated the cysteine sulfinate decarboxylase activity. These results indicate that the availability of sulfur amino acid(s) especially cysteine appears to play a role in determining the flux of cysteine between cysteine catabolism and GSH synthesis.

Treatment of cicatricial lagophthalmos: very small orbicularis oculi muscle pedicled skin flap.

There are many methods for the correction of cicatricial lagophthalmos. Skin and soft tissue should be used to reconstruct the defect after release of tension. We used a very small orbicularis oculi muscle pedicled skin flap for the correction of mild to moderate degrees of cicatricial lagophthalmos in six cases. A small skin island flap from near the skin defect, pedicled on the orbicularis oculi muscle, was transposed to fill the defect. The length of the skin flap ranged from 5 mm to 11 mm. With a follow-up period ranging from 6 months to 18 months (mean: 11 months),we obtained satisfactory functional and aesthetic results. The eyelid closure was much improved and the discomfort was relieved. All the flaps survived without any healing problems. This small orbicularis oculi myocutaneous flap is very reliable and has a wide range of motion. This technique can be applied to eyelid-skin defects of various causes.

A new classification of palatal fracture and an algorithm to establish a treatment plan.

Palatal fractures have previously been classified according to the anatomic location of the fracture line, which is helpful for understanding the types of palatal fracture, but which is insufficient for helping the surgeon to decide which fracture to open and how to do so. The purpose of this study was to aid in the establishment of a precise treatment plan by determining the surgical approach and the types of stabilization that should be used for different types of palatal fracture. In a retrospective review of 136 consecutive Le Fort maxillary fractures over 6 years, 18 patients (13.2 percent) with palatal fractures were analyzed. The principle of open reduction and internal fixation was applied to all the patients. In six patients (33 percent), exploration and fixation was done in the palatal surface. Eight patients (44 percent) needed an extended period of immobilization (4 to 6 weeks). No major complications were observed during the follow-up period. An algorithm was devised to help establish a proper treatment plan, and palatal fractures were classified into four types: closed reduction, anterior treatment, anterior and palatal treatment, and combined. The key elements considered in deciding the treatment principle and the classification of a palatal fracture were the possibility of closed reduction, surgical exposure, site of rigid fixation, and stability of fractured segments after rigid fixation. The outcome of reconstruction and the postoperative course differed depending on the type of palatal fracture. This classification scheme provided an easy and simple way to establish a treatment plan and was helpful in learning the treatment principles of palatal fracture.