Copy number elevation of 22q11.2 genes arrests the developmental maturation of working memory capacity and adult hippocampal neurogenesis.

Working memory capacity, a critical component of executive function, expands developmentally from childhood through adulthood. Anomalies in this developmental process are seen in individuals with autism spectrum disorder (ASD), schizophrenia and intellectual disabilities (ID), implicating this atypical process in the trajectory of developmental neuropsychiatric disorders. However, the cellular and neuronal substrates underlying this process are not understood. Duplication and triplication of copy number variants of 22q11.2 are consistently and robustly associated with cognitive deficits of ASD and ID in humans, and overexpression of small 22q11.2 segments recapitulates dimensional aspects of developmental neuropsychiatric disorders in mice. We capitalized on these two lines of evidence to delve into the cellular substrates for this atypical development of working memory. Using a region- and cell-type-selective gene expression approach, we demonstrated that copy number elevations of catechol-O-methyl-transferase (COMT) or Tbx1, two genes encoded in the two small 22q11.2 segments, in adult neural stem/progenitor cells in the hippocampus prevents the developmental maturation of working memory capacity in mice. Moreover, copy number elevations of COMT or Tbx1 reduced the proliferation of adult neural stem/progenitor cells in a cell-autonomous manner in vitro and migration of their progenies in the hippocampus granular layer in vivo. Our data provide evidence for the novel hypothesis that copy number elevations of these 22q11.2 genes alter the developmental trajectory of working memory capacity via suboptimal adult neurogenesis in the hippocampus.

Report From the American Society of Transplantation Conference on Donor Heart Selection in Adult Cardiac Transplantation in the United States.

Cardiac transplantation remains the only definitive treatment for end-stage heart failure. Transplantation rates are limited by a shortage of donor hearts. This shortage is magnified because many hearts are discarded because of strict selection criteria and concern for regulatory reprimand for less-than-optimal posttransplant outcomes. There is no standardized approach to donor selection despite proposals to liberalize acceptance criteria. A donor heart selection conference was organized to facilitate discussion and generate ideas for future research. The event was attended by 66 participants from 41 centers with considerable experience in cardiac donor selection. There were state-of-the-art presentations on donor selection, with subsequent breakout sessions on standardizing the process and increasing utilization of donor hearts. Participants debated misconceptions and established agreement on donor and recipient risk factors for donor selection and identified the components necessary for a future donor risk score. Ideas for future initiatives include modification of regulatory practices to consider extended criteria donors when evaluating outcomes and prospective studies aimed at identifying the factors leading to nonacceptance of available donor hearts. With agreement on the most important donor and recipient risk factors, it is anticipated that a consistent approach to donor selection will improve rates of heart transplantation.

Efficacy and safety of rebamipide liquid for chemoradiotherapy-induced oral mucositis in patients with head and neck cancer: a multicenter, randomized, double-blind, placebo-controlled, parallel-group phase II study.

BACKGROUND: Recent preclinical and phase I studies have reported that rebamipide decreased the severity of chemoradiotherapy-induced oral mucositis in patients with oral cancer. This placebo-controlled randomized phase II study assessed the clinical benefit of rebamipide in reducing the incidence of severe chemoradiotherapy-induced oral mucositis in patients with head and neck cancer (HNC). METHODS: Patients aged 20-75 years with HNC who were scheduled to receive chemoradiotherapy were enrolled. Patients were randomized to receive rebamipide 2% liquid, rebamipide 4% liquid, or placebo. The primary endpoint was the incidence of grade ≥ 3 oral mucositis determined by clinical examination and assessed by central review according to the Common Terminology Criteria of Adverse Events version 3.0. Secondary endpoints were the time to onset of grade ≥ 3 oral mucositis and the incidence of functional impairment (grade ≥ 3) based on the evaluation by the Oral Mucositis Evaluation Committee. RESULTS: From April 2014 to August 2015, 97 patients with HNC were enrolled, of whom 94 received treatment. The incidence of grade ≥ 3 oral mucositis was 29% and 25% in the rebamipide 2% and 4% groups, respectively, compared with 39% in the placebo group. The proportion of patients who did not develop grade ≥ 3 oral mucositis by day 50 of treatment was 57.9% in the placebo group, whereas the proportion was 68.0% in the rebamipide 2% group and 71.3% in the rebamipide 4% group. The incidences of adverse events potentially related to the study drug were 16%, 26%, and 13% in the placebo, rebamipide 2%, and rebamipide 4% groups, respectively. There was no significant difference in treatment compliance among the groups. CONCLUSIONS: The present phase II study suggests that mouth washing with rebamipide may be effective and safe for patients with HNC receiving chemoradiotherapy, and 4% liquid is the optimal dose of rebamipide. TRIAL REGISTRATION: ClinicalTrials.gov under the identifier NCT02085460 (the date of trial registration: March 11, 2014).

Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes.

Low concentrations (0.11-1.7 microg ml(-1)) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modified by amino groups, when added with nerve growth factor (NGF), promoted outgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and rat pheochromocytoma cell line PC12h cells in culture media. The quantity of active extracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 microg ml(-1) CNTs and NGF than that with NGF alone. CNTs increased the number of cells with neurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERK signaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after the addition of CNTs to the culture medium. These results demonstrate that CNTs may stimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs are biocompatible and are promising candidates for biological applications and devices.

Wholly vascularized millimeter-sized engineered tissues by cell-sized microscaffolds.

The in vitro fabrication of wholly vascularized millimeter-sized engineered tissues is still a key challenge in the tissue engineering field. Recently we reported a unique approach 'sedimentary culture' using a collagen microfiber (CMF) to fabricate large-scale engineered tissues. The millimeter-sized tissues with high extracellular matrix (ECM) density were easily obtained by centrifugation of cells and CMFs and subsequent cultivation because the CMFs acted as a micrometer-sized scaffold. However, cell distribution in the obtained tissues was not homogeneous because of the different sedimentation velocity of the cells and CMFs because of their size difference. Here we report the fabrication of wholly vascularized millimeter-sized engineered tissues using cell-sized CMFs. To avoid dissolving, vacuum drying was performed at 200 °C for 24 h for thermal crosslinking of primary amine groups of type I collagen. The 200- and 20-µm-sized CMFs (CMF-200 and CMF-20) were obtained by homogenization and subsequent sonication of the crosslinked collagen. Interestingly, the CMF-20 indicated a similar sedimentation velocity with cells because of their same size range, thus uniform millimeter-sized tissue with homogeneous cell distribution was fabricated by the sedimentary culture method. To form a whole blood capillary structure in the tissues, fibronectin (FN) was adsorbed on the surface of CMF-20 to stimulate endothelial cell migration. The distribution of the blood capillary network in 1.6-mm-sized tissues was markedly improved by FN-adsorbed CMF-20 (FN-CMF-20). Sedimentary culture using FN-CMF-20 will create new opportunities in tissue engineering for the in vitro fabrication of wholly vascularized millimeter-sized engineered tissues.

Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.

Cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway.

Nitric oxide (NO) is a novel biologic messenger with diverse effects but its role in organ transplantation remains poorly understood. Using a porphyrinic microsensor, the first direct measurements of coronary vascular and endocardial NO production were made. NO was measured directly in the effluent of preserved, heterotopically transplanted rat hearts stimulated with L-arginine and bradykinin; NO concentrations fell from 2.1 +/- 0.4 microM for freshly explanted hearts to 0.7 +/- 0.2 and 0.2 +/- 0.08 microM for hearts preserved for 19 and 38 h, respectively. NO levels were increased by SOD, suggesting a role for superoxide-mediated destruction of NO. Consistent with these data, addition of the NO donor nitroglycerin (NTG) to a balanced salt preservation solution enhanced graft survival in a time- and dose-dependent manner, with 92% of hearts supplemented with NTG surviving 12 h of preservation versus only 17% in its absence. NTG similarly enhanced preservation of hearts stored in University of Wisconsin solution, the clinical standard for preservation. Other stimulators of the NO pathway, including nitroprusside, L-arginine, or 8-bromoguanosine 3',5' monophosphate, also enhanced graft survival, whereas the competitive NO synthase antagonist NG-monomethyl-L-arginine was associated with poor preservation. Likely mechanisms whereby supplementation of the NO pathway enhanced preservation included increased blood flow to the reperfused graft and decreased graft leukostasis. NO was also measured in endothelial cells subjected to hypoxia/reoxygenation and detected based on its ability to inhibit thrombin-mediated platelet aggregation and serotonin release. NO became undetectable in endothelial cells exposed to hypoxia followed by reoxygenation and was restored to normoxic levels on addition of SOD. These studies suggest that the NO pathway fails during preservation/transplantation because of formation of oxygen free radicals during reperfusion, which quench available NO. Augmentation of NO/cGMP-dependent mechanisms enhances vascular function after ischemia and reperfusion and provides a new strategy for transplantation of vascular organs.

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Antisense intercellular adhesion molecule-1 (ICAM-1) oligodeoxyribonucleotide delivered during organ preservation inhibits posttransplant ICAM-1 expression and reduces primary lung isograft failure.

Transiently increased expression of leukocyte adhesion receptors after lung preservation contributes to early graft demise by recruiting leukocytes, activating complement, and causing microcirculatory stasis. We hypothesized that inhibiting intercellular adhesion molecule-1 (ICAM-1) expression even briefly may significantly improve lung graft function and that the preservation period might provide a unique window to deliver a therapeutic pulse of antisense oligonucleotide ICAM-1 to inhibit ICAM-1 expression after transplantation. Interleukin-1beta-treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3'-untranslated region of rat ICAM-1 demonstrated an oligonucleotide dose-dependent reduction in ICAM-1 expression. Using a cationic liposomal carrier, this same antisense oligonucleotide (but not the sense control) instilled into the pulmonary vasculature at the time of preservation reduced subsequent graft ICAM-1 expression and graft leukostasis and markedly improved oxygenation, pulmonary blood flow, and graft survival. These experiments demonstrate that the preservation period presents a window during which to target an anti-ICAM-1 expression strategy to inhibit early adhesion receptor expression and improve functional outcome after lung transplantation.

cAMP pulse during preservation inhibits the late development of cardiac isograft and allograft vasculopathy.

The causes of transplant-associated coronary artery disease remain obscure, and there is no known treatment. Preservation injury of murine heterotopic vascularized cardiac isografts caused a small, albeit significant, increase in neointimal formation; preservation injury of allografts markedly increased both the incidence and severity of transplant-associated coronary artery disease. As cAMP is an important vascular homeostatic mediator the levels of which decline during organ preservation, buttressing cAMP levels solely during initial preservation both improved acute allograft function and reduced the severity of transplant-associated coronary artery disease in grafts examined 2 months later. Inhibiting the cAMP-dependent protein kinase abrogated these beneficial effects. cAMP treatment was associated with an early reduction in leukocyte infiltration and a reciprocal decrease in superoxide and increase in NO levels. These data indicate that alloantigen-independent injury to the graft, which occurs at the time of cardiac preservation, can set in motion pathological vascular events that are manifest months later. Furthermore, a cAMP pulse during cardiac preservation reduces the incidence and severity of transplant-associated coronary artery disease.

Necrotizing effect of ethanedimethanesulfonate on spontaneously occurring Leydig cell tumors in old F344 rats.

Thirty 18-month-old male F344/DuCrj rats were divided into the following groups: 10 untreated controls; eight vehicle-injected controls; and 12 ethanedimethanesulfonate (EDS)-injected rats. Untreated controls were killed immediately to check for testicular tumor incidence. In rats of the test group, a 75-mg/kg dose of EDS dissolved in dimethyl sulfoxide:water (1:3) was injected i.p. At intervals of 1, 2, 3, and 10 days after injection, two vehicle-injected control rats and three EDS-injected rats were sacrificed, and the testes were fixed by vascular perfusion. The midsagittal sections of all the fixed testes were examined to determine the incidence of macroscopic Leydig cell tumors, and some tumor tissues of the injection-treated groups were also investigated ultrastructurally. In 28 of 30 animals, a total of 78 Leydig cell tumors could be distinguished. Extensive and severe necrotic alterations accompanying fresh, multiple hemorrhages in early stages and reparative changes in later stages could be observed in a total of 78% of the 32 tumors examined from the EDS-injected group. The tumor cells exhibited ultrastructurally degenerative changes such as chromatin condensation and cytoplasmic vacuolation from 1 day after EDS injection. Therefore, EDS may be a necrotic agent for rat Leydig cell tumor.

Comparison of right and left ventricular responses to left ventricular assist device support in patients with severe heart failure: a primary role of mechanical unloading underlying reverse remodeling.

BACKGROUND: Left ventricular assist devices (LVAD) reverse ventricular, myocardial, and systemic abnormalities characteristic of severe heart failure (reverse remodeling). The relative contributions of hemodynamic unloading and normalized biochemical milieu to reverse remodeling are unknown. METHODS AND RESULTS: Structural and functional characteristics were measured from 53 hearts of patients undergoing transplantation without LVAD support (medical support) and 33 hearts from patients receiving a median of 46 days of LVAD support (range, 8 to 360 days). Compared with medical support alone, patients receiving LVAD support for >/=30 days had higher central venous pressures (11+/-6 versus 8+/-5 mm Hg, P=0.04), lower pulmonary artery diastolic pressures (14+/-9 versus 21+/-9 mm Hg, P=0.01), and higher cardiac outputs (5.1+/-1.6 versus 3.7+/-1.0 L/min, P<0.001). In LVAD versus transplantation hearts, V(30) (ex vivo volume yielding ventricular pressure of 30 mm Hg) was decreased in the left ventricle (LV) (179+/-75 versus 261+/-118 mL, P=0.005) but not in the right ventricle (RV) (140+/-59 versus 148+/-52 mL, P=NS). LV myocyte diameter decreased more significantly after LVAD support (17%, P=0.05) than in the RV (11%, P=NS). Compared with transplantation, LVAD support increased normalized SERCA2a content in the LV (0.51+/-0.26 versus 1.04+/-0.34, P<0.001) but not in the RV (0.48+/-34 versus 0.67+/-0.55, P=NS). Finally, LVAD support improved force-frequency relations of isolated superfused LV trabeculae (P=0.01) but not RV trabeculae. CONCLUSIONS: Reduction of hemodynamic load is a primary factor underlying several important features of reverse remodeling. These findings do not preclude a possible primary role of neurohormonal factors underlying other facets of reverse remodeling during LVAD support.

Case of trigger finger related to an intertendinous connection between the flexor tendons.

We report a rare cause of trigger finger related to an anatomical variation of an intertendinous connection between the flexor digitorum superficialis and flexor digitorum profundus tendons in the palmar region.

Cardiac Donor Risk Factors Predictive of Short-Term Heart Transplant Recipient Mortality: An Analysis of the United Network for Organ Sharing Database.

INTRODUCTION: To address the shortage of donor hearts for transplantation, there is significant interest in liberalizing donor acceptance criteria. Therefore, the aim of this study was to evaluate cardiac donor characteristics from the United Network for Organ Sharing (UNOS) database to determine their impact on posttransplantation recipient outcomes. METHODS: Adult (≥18 years) patients undergoing heart transplantation from July 1, 2004, to December 31, 2012, in the UNOS Standard Transplant Analysis and Research (STAR) database were reviewed. Patients were stratified by 1-year posttransplantation status; survivors (group S, n = 13,643) and patients who died or underwent cardiac retransplantation at 1-year follow-up (group NS/R = 1785). Thirty-three specific donor variables were collected for each recipient, and independent donor predictors of recipient death or retransplantation at 1 year were determined using multivariable logistic regression analysis. RESULTS: Overall 1-year survival for the entire cohort was 88.4%. Mean donor age was 31.5 ± 11.9 years, and 72% were male. On multivariable logistic regression analysis, donor age >40 years (odds ratio [OR] 1.44, 95% confidence interval [CI] 1.27 to 1.64), graft ischemic time >3 hours (OR 1.32, 1.16 to 1.51), and the use of cardioplegia (OR 1.17, 1.01 to 1.35) or Celsior (OR 1.21, 1.06 to 1.38) preservative solution were significant predictors of recipient death or retransplantation at 1 year posttransplantation. Male donor sex (OR 0.83, 0.74 to 0.93) and the use of antihypertensive agents (OR 0.88, 0.77 to 1.00) or insulin (OR 0.84, 0.76 to 0.94) were protective from adverse outcomes at 1 year. CONCLUSIONS: These data suggest that donors who are older, female, or have a long projected ischemic time pose greater risk to heart transplant recipients in the short term. Additionally, certain components of donor management protocols, including antihypertensive and insulin administration, may be protective to recipients.

Innervation of the cerebral veins as compared with the cerebral arteries: a histochemical and electron microscopic study.

The distribution of nerve fibers in the cerebral veins was studied by catecholamine fluorescence simultaneously with acetylcholinesterase (AChE) histochemistry. A comparison of nerve fibers in the cerebral arteries was made. The ultrastructure of terminal boutons in the veins fixed with potassium permanganate was also studied. In the adventitia of the cerebral artery, green fluorescent aminergic fibers and brownish AChE-reactive (probably cholinergic) fibers were observed. In contrast, the cerebral venous system showed no AChE-positive fibers. Catecholamine fluorescent varicose fibers were detected in the dural sinus, the internal cerebral vein, and the superficial vein of Labbé. The highest density of aminergic fibers was found in the dural sinus and the second highest in the internal cerebral vein. Most of the terminal boutons in the adventitia of the cerebral veins were found adjacent to a muscle-like cell and showed only cored vesicles under electron microscopy. Results of our study suggest that the cerebral venous system has a neurogenic innervation, mainly from aminergic fibers, which is different from the neurogenic supply to the cerebral arterial system.

Immunohistochemical demonstration of vasopressin nerve fibers in the cerebral artery.

Vasopressin-immunoreactive nerve fibers were demonstrated in the cerebral pial arteries by peroxidase immunohistochemistry. In the large pial artery (proximal part of the middle cerebral artery), they ran longitudinally to the long axis of the vessel. They ran in a spiral pattern in the distal part of the middle cerebral artery. Even in small arteries, vasopressin nerve fibers were found arranged in a longitudinal fashion. The present morphological data suggest that vasopressin nerve fibers in the cerebral artery may play a role in cerebral circulation.

A light and electron microscopic immunohistochemical study of vasoactive intestinal polypeptide- and substance P-containing nerve fibers along the cerebral blood vessels: comparison with aminergic and cholinergic nerve fibers.

Vasoactive intestinal polypeptide (VIP)- and substance P-containing nerve fibers were observed in the cerebral blood vessels using an immunohistochemical technique. VIP-containing nerve fibers distributed in a spiral pattern, similar to that of muscle cells. Under electron microscopic observation, VIP-immunoreactive terminals lay close to a muscle cell in the inner layer of the adventitia. In contrast, substance P-containing nerve fibers showed a meshwork pattern in the outer layer of the adventitia. Using both acetylcholinesterase (AChE) staining and VIP immunohistochemistry, AChE-positive and VIP-immunoreactive nerve fibers revealed almost the same distribution in the same specimen. The present data suggest that VIP-containing nerve fibers may play a role in the smooth muscle control of the blood vessels, whereas substance P-containing nerve fibers may not take part in muscle control.