Ko Hosokawa

Bone marrow cells differentiate into wound myofibroblasts and accelerate the healing of wounds with exposed bones when combined with an occlusive dressing

Background  The usefulness of bone marrow cells in accelerating wound healing has not been evaluated despite increasing evidence that bone marrow contains mesenchymal stem cells that have multipotentiality to differentiate into various types of cells after they enter the microenvironment of a specific tissue (niche).

Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays.

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post‐injured sciatic nerve which was enriched in non‐myelinating Schwann cells using cDNA microarrays. The number of up‐ and down‐regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up‐regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down‐regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co‐receptor for glial cell line‐derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.

Activation of Rho in the injured axons following spinal cord injury

Axons of the adult central nervous system have very limited ability to regenerate after injury. This inability may be, at least partly, attributable to myelin‐derived proteins, such as myelin‐associated glycoprotein, Nogo and oligodendrocyte myelin glycoprotein. Recent evidence suggests that these proteins inhibit neurite outgrowth by activation of Rho through the neurotrophin receptor p75NTR/Nogo receptor complex. Despite rapidly growing knowledge on these signals at the molecular level, it remained to be determined whether Rho is activated after injury to the central nervous system. To assess this question, we establish a new method to visualize endogenous Rho activity in situ. After treatment of cerebellar granular neurons with the Nogo peptide in vitro, Rho is spatially activated and colocalizes with p75NTR. Following spinal cord injury in vivo, massive activation of Rho is observed in the injured neurites. Spatial regulation of Rho activity may be necessary for axonal regulation by the inhibitory cues.

Embolization of High Flow Arteriovenous Malformations: Experience with Use of Superabsorbent Polymer Microspheres

PURPOSE To determine efficacy, safety, and requirements for adjunctive embolization or surgery in the treatment of symptomatic arteriovenous malformations (AVMs) with superabsorbent polymer microsphere (SAP-MS) particles. MATERIALS AND METHODS SAP-MS particles (sodium acrylate and vinyl alcohol copolymer) are nonbiodegradable spheres with a precisely calibrated diameter. SAP-MS particles swell by absorbing fluids and become soft and deformable. Twenty-five patients (16 men, nine women; mean age, 32 y; range 12-66 y) with symptomatic facial (n = 5), upper- (n = 8) and lower- (n = 12) extremity AVMs were treated primarily (n = 23) or preoperatively (n = 2) by transarterial embolization (TAE) treatment with use of SAP-MS particles. Direct puncture embolization (DPE; n = 4) and/or surgical intervention (n = 5; ie, skin graft, resection, or amputation) were required. Surgical specimens from the resected (n = 2) and the amputated (n = 2) patients were evaluated histologically. Follow-up study, including clinical findings and imaging studies, was performed at intervals ranging from 3 months to 1 year. Clinical outcome was evaluated retrospectively, depending on the subjective improvement of symptoms and signs, according to the medical records. RESULTS Seventy-two TAEs (range, 1-11; mean, 2.8) and 12 DPEs (range, 1-3; mean, 2.4) were performed during the mean follow-up period of 38 months (range, 7-110 mo). Twenty patients (80%) experienced symptom improvement by embolotherapy alone (n = 17) or in combination with surgery (n = 3). One lip and two finger AVMs were totally removed by surgical excision or amputation after TAE treatment. In diffuse upper- (n = 1) and lower- (n = 1) extremity AVMs, the symptoms were uncontrolled. No nerve injury or skin necrosis was observed after TAE treatment with SAP-MS particles. Mucosal necrosis was induced by DPE with ethanol in one patient. Histologically, SAP-MS particles penetrated intralesional vessels and conformed to the vessel lumen, resulting in tight vessel occlusion. Minimal perivascular reaction was observed. CONCLUSION SAP-MS particles were used safely in TAE treatment of AVM. TAE treatment with use of SAP-MS particles was suitable for certain symptomatic AVMs, but diffuse AVMs remain a challenge and a combination of alternative methods will be necessary for further strategy.

Glial differentiation of human adipose-derived stem cells: implications for cell-based transplantation therapy.

Increasing evidence has shown that adipose-derived stem cells (ASCs) could transdifferentiate into Schwann cell (SC)-like cells to enhance nerve regeneration, suggesting potential new cell-based transplantation therapy for peripheral nerve injuries and neurodegenerative disorders. For the implementation of these results to the clinical setting, it is of great importance to establish the differentiation of human ASCs (hASCs) into a SC phenotype. In this study, we studied hASCs obtained from subcutaneous fat tissue of healthy donors. By a mixture of glial growth factors we differentiated them into Schwann cell-like cells (dhASCs). We then assessed their ability to act as Schwann cells in vitro and in vivo and also compared them with primary human Schwann cells (hSCs). Enzyme-linked immunosorbent assay showed that dhASCs secreted brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF) at a comparable level, and glial cell-derived neurotrophic factor (GDNF) at a level even higher than hSCs, whereas undifferentiated hASCs (uhASCs) secreted low levels of these neurotrophic factors. In co-culture with NG108-15 neuronal cells we found that both dhASCs and hSCs significantly increased the percentage of cells with neurites, the neurite length, and the number of neurites per neuron, whereas uhASCs increased only the percentage of cells with neurites. Finally, we transplanted green fluorescent protein (GFP)-labeled hASCs into the crushed tibial nerve of athymic nude rats. The transplanted hASCs showed a close association with PGP9.5-positive axons and myelin basic protein (MBP)-positive myelin at 8weeks after transplantation. Quantitative analysis revealed that dhASCs transplantation resulted in significantly improved survival and myelin formation rates (a 7-fold and a 10-fold increase, respectively) as compared with uhASCs transplantation. These findings suggest that hASCs took part in supporting and myelinating regenerating axons, and thus have achieved full glial differentiation in vivo. In conclusion, hASCs can differentiate into SC-like cells that possess a potent capacity to secrete neurotrophic factors as well as to form myelin in vivo. These findings make hASCs an interesting prospect for cell-based transplantation therapy for various peripheral nerve disorders.

bFGF regulates PI3-kinase-Rac1-JNK pathway and promotes fibroblast migration in wound healing.

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration.

Postoperative seroma formation in breast reconstruction with latissimus dorsi flaps: a retrospective study of 174 consecutive cases.

The latissimus dorsi flap has been widely used in breast reconstruction surgery. Despite its potential advantages such as low donor morbidity and vascular reliability, the complication of donor-site seroma formation frequently occurs. Consecutive 174 patients who underwent breast reconstruction with the latissimus dorsi flap from 2001 to 2006 were retrospectively reviewed. The age, body mass index (BMI), smoking history, timing of reconstruction, type of breast surgery and nodal dissection, and several other intraoperative data were analyzed. The overall incidence of postoperative seroma was 21%. Increased age (>50 years) and obesity (BMI >23 kg/m2) were significant risk factors for seroma formation (P = 0.02 and 0.004, respectively). The patients who underwent skin-sparing mastectomy or modified radical mastectomy had higher incidence of seroma formation (28% and 33%, respectively) as compared with those who had breast-conservative surgery (11%). A significant correlation was found between the type of breast surgery and the incidence of seroma (P = 0.04). The type of nodal dissection did not affect the incidence of postoperative seroma (P = 0.66). We concluded that increased age, obesity, and invasive breast surgery are risk factors for donor-site seroma formation after breast reconstruction with the latissimus dorsi flap. Close attention should be paid to prevent development of postoperative seroma when operating on such high-risk patients.

Vascular anatomy of the anterolateral thigh flap.

Arterial and venous anatomy and their relation to the anterolateral thigh flap were examined in 10 specimens of six fresh cadavers in which radiopaque materials were injected into both the arterial and venous systems. Territories and positions of individual perforating arteries were measured, and the venous drainage pathway of the flap was analyzed. All specimens were radiographed stereoscopically to observe the three-dimensional structure of the arteries and veins. The territory of each perforating artery was smaller than expected. Most of the venous blood that had perfused the dermis was considered to pool in a polygonal venous network located in the skin layer and to enter the descending branch of the lateral circumflex femoral artery through large descending veins. The venous territories were considered different from the arterial territories. The findings in this study suggest that the design of the anterolateral thigh flap should be based on the venous architecture rather than on the arterial architecture and that the flap survival rate might be improved if thinning is performed appropriately.

Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function.

Aryl hydrocarbon receptor nuclear translocator (ARNT), a transcription factor of the Per/AHR/ARNT/Sim family, regulates gene expression in response to environmental stimuli including xenobiotics and hypoxia. To examine its role in the epidermis, the Cre-loxP system was used to disrupt the Arnt gene in a keratinocyte-specific manner. Gene-targeted, newborn mice with almost normal appearance died neonatally of severe dehydration caused by water loss. Histology showed small changes in the architecture of cornified layers, with apparently preserved intercorneocyte lamellar structures responsible for the skin barrier function. In contrast, HPLC/ion-trap mass spectrometry revealed significant alterations in the compositions of ceramides, the major components of the lamellae. The murine epidermal ceramides normally contain 4-sphingenine and 4-hydroxysphinganine. In Arnt-null epidermis, 4-sphingenine was largely replaced by sphinganine and the amounts of ceramides with 4-hydroxysphinganine were greatly decreased, suggesting deficiency of dihydroceramide desaturases that catalyze the formation of both 4-sphingenyl and 4-hydroxysphinganyl moieties. A desaturase isoenzyme, DES-1, prefers desaturation, but DES-2 catalyzes both reactions to a similar extent. Transcript levels of Des-2, but not Des-1, were considerably decreased in cultured keratinocytes from Arnt-null epidermis. These results indicate that proper ceramide compositions through 4-desaturation regulated by ARNT are crucial for maintaining the epidermal barrier function.

The Animal Model of Spinal Cord Injury as an Experimental Pain Model

Pain, which remains largely unsolved, is one of the most crucial problems for spinal cord injury patients. Due to sensory problems, as well as motor dysfunctions, spinal cord injury research has proven to be complex and difficult. Furthermore, many types of pain are associated with spinal cord injury, such as neuropathic, visceral, and musculoskeletal pain. Many animal models of spinal cord injury exist to emulate clinical situations, which could help to determine common mechanisms of pathology. However, results can be easily misunderstood and falsely interpreted. Therefore, it is important to fully understand the symptoms of human spinal cord injury, as well as the various spinal cord injury models and the possible pathologies. The present paper summarizes results from animal models of spinal cord injury, as well as the most effective use of these models.