Diagnosis of Patients Presenting with Vertigo, Headache, and Epileptic Seizure: Evaluating Vestibular Patients by Using Cervical Vestibular Evoked Myogenic Potential and Auditory Middle Latency Responses in the Clinical Setting.

Migraine and vertigo are common complaints seen in clinical practice, and in a few such cases, we also find epileptic manifestations, including migraine-triggered seizures. Currently, patients presenting with vertigo and headache are diagnosed according to established diagnostic criteria for Meniere's disease, vestibular migraine, or vestibular migraine/Meniere's disease overlapping syndrome. In addition to using those diagnostic criteria and the patient's history, cervical vestibular evoked myogenic potential and auditory middle latency responses are useful tools to better understand the physiological background of these patients and also to confirm the diagnosis. Here we report 2 cases: 1 of vestibular migraine/ Meniere's disease overlapping syndrome and 1 of vestibular migraine with epileptic manifestations. Each patient showed potentiation (lack of habituation) in auditory middle latency response, and each showed endolymphatic hydrops in cervical vestibular evoked myogenic potential. The potentiation in auditory middle latency response might be attributable to neuronal hyperexcitability in those patients with migraine or epilepsy, and neurogenic inflammation caused by migraine episodes might affect inner ear function.

Cervical vestibular evoked myogenic potential with chirp sounds.

BACKGROUND: There are only a few reports concerning cervical vestibular evoked myogenic potential (cVEMP) using chirp sound, and clinical indications/advantages of it are still unclear. OBJECTIVE: To compare cVEMP using CE-chirp LS® with cVEMP using 500 Hz and 1000 Hz tone bursts (TB) and to investigate clinical indications/advantages of CE-chirp LS® for recording cVEMP. METHODS: Sixteen patients with vestibular disorders (2men and 14 women) (18∼62, mean 42.9 years of age) were enrolled in this study. Participants underwent cVEMP testing using 500 Hz and 1000 Hz tone bursts (TB) and CE-chirp LS®. Response rate of P1-N1, corrected/normalized amplitude of P1-N1, latencies of P1 and N1, asymmetry ratio, and correlation of P1 latency to SLOPE in tuning property test (an index of endolymphatic hydrops) were compared. RESULTS: Corrected/normalized amplitude of P1-N1 to CE-chirp LS® was smaller than corrected/normalized amplitude of P1-N1 to 500 Hz TB. Peak latencies to CE-chirp LS® were the shortest among the 3 types of stimulation. EH-positive ears according to the tuning property test had tendency of prolonged P1 latencies to CE-chirp LS®. CONCLUSION: CE-chirp LS® is applicable for recording cVEMP with a similar diagnostic accuracy to TB. Prolongation of P1 latency in CE-chirp LS® might be an indicator of endolymphatic hydrops in the saccule.

Effectiveness of probiotic therapy for the prevention of relapse in patients with inactive ulcerative colitis.

AIM: To evaluate the effectiveness of probiotic therapy for suppressing relapse in patients with inactive ulcerative colitis (UC). METHODS: Bio-Three tablets, each containing 2 mg of lactomin (Streptococcus faecalis T-110), 10 mg of Clostridium butyricum TO-A, and 10 mg of Bacillus mesentericus TO-A, were used as probiotic therapy. Sixty outpatients with UC in remission were randomly assigned to receive 9 Bio-Three tablets/day (Bio-Three group) or 9 placebo tablets/day (placebo group) for 12 mo in addition to their ongoing medications. Clinical symptoms were evaluated monthly or on the exacerbation of symptoms or need for additional medication. Fecal samples were collected to analyze bacterial DNA at baseline and 3-mo intervals. Terminal restriction fragment length polymorphism and cluster analyses were done to examine bacterial components of the fecal microflora. RESULTS: Forty-six patients, 23 in each group, completed the study, and 14 were excluded. The relapse rates in the Bio-Three and placebo groups were respectively 0.0% vs 17.4% at 3 mo (P = 0.036), 8.7% vs 26.1% at 6 mo (P = 0.119), and 21.7% vs 34.8% (P = 0.326) at 9 mo. At 12 mo, the remission rate was 69.5% in the Bio-Three group and 56.6% in the placebo group (P = 0.248). On cluster analysis of fecal flora, 7 patients belonged to cluster I, 32 to cluster II, and 7 to cluster III. CONCLUSION: Probiotics may be effective for maintaining clinical remission in patients with quiescent UC, especially those who belong to cluster I on fecal bacterial analysis.

Damaged utricular function clarified by oVEMP in patients with benign paroxysmal positional vertigo.

CONCLUSION: Utricular dysfunction in patients with posterior canal benign paroxysmal positional vertigo (pBPPV) was supported by findings for ocular vestibular evoked myogenic potential (oVEMP). OBJECTIVE: To evaluate the utricular and saccular function in patients with pBPPV. METHODS: This study focused on 12 patients definitively diagnosed with pBPPV showing typical nystagmus by Dix-Hallpike maneuver and 12 controls. In these subjects, oVEMPs and cVEMPs to air-conducted 500 Hz tone burst (125 dB SPL) were measured. The patients also underwent caloric tests. RESULTS: More of the patients with pBPPV showed abnormal responses in oVEMPs by stimulation on their affected side than the controls, while the results of cVEMPs showed no significant differences between pBPPV patients and controls. The abnormal results for oVEMPs on the affected side showed a higher percentage than those for cVEMPs and caloric tests in pBPPV patients. There was no significant association between any of the tests. These findings support the possibility that oVEMP reflects the specific abnormal condition in pBPPV, i.e. that the urticular function in pBPPV patients was highly damaged.

Association of air-conducted sound oVEMP findings with cVEMP and caloric test findings in patients with unilateral peripheral vestibular disorders.

CONCLUSION: This study showed that the ocular vestibular evoked myogenic potential (oVEMP) in response to air-conducted sound (ACS) reflects functions of different parts of the vestibular labyrinth from cervical VEMP (cVEMP). OBJECTIVE: To determine whether the origin of the vestibular end organs of the oVEMP in response to ACS (500 Hz tone bursts) is the same as that of cVEMP. METHODS: Twenty patients definitively diagnosed with unilateral Meniere's disease (MD), 6 patients with unilateral vestibular neuritis (VN), and 7 healthy subjects were enrolled. In these subjects, the oVEMP and cVEMP to air-conducted 500 Hz tone bursts (125 dBSPL) were measured. The patients also underwent caloric tests. RESULTS: The MD patients did not show a significant association between their ACS oVEMP findings and ACS cVEMP findings but there was an association of ACS oVEMP findings with caloric test findings. When the MD patients were classified into four stages based on their hearing levels, the patients showed abnormal findings at earlier stages on ACS cVEMP than on other tests. While all six VN patients showed abnormal findings on ACS oVEMP and caloric tests, only two patients showed abnormal ACS cVEMPs. These findings support the hypothesis that the oVEMP in response to ACS predominantly reflects utricular functions while ACS cVEMP reflects saccular functions.

A neurospheroid network-stamping method for neural transplantation to the brain.

Neural transplantation therapy using neural stem cells has received as potential treatments for neurodegenerative diseases. Indeed, this therapy is thought to be effective for replacement of degenerating neurons in restricted anatomical region. However, because injected neural stem cells integrate randomly into the host neural network, another approach is needed to establish a neural pathway between selective areas of the brain or treat widespread degeneration across multiple brain regions. One of the promising approaches might be a therapy using pre-made neural network in vitro by the tissue engineering technique. In this study, we engineered a three-dimensional (3D) tissue with a neuronal network that can be easily manipulated and transplanted onto the host brain tissue in vivo. A polydimethylsiloxane microchamber array facilitated the formation of multiple neurospheroids, which in turn interconnected via neuronal processes to form a centimeter-sized neurospheroid network (NSN). The NSN was transferable onto the cortical surface of the brain without damage of the neuronal network. After transfer onto the cortical tissue, the NSN showed neural activity for more than 8 days. Moreover, neurons of the transplanted NSN extended their axons into the host cortical tissue and established synaptic connections with host neurons. Our findings suggest that this method could lay the foundation for treating severe degenerative brain disease.

Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.

Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 degrees C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation.

Monodisperse cell-encapsulating peptide microgel beads for 3D cell culture.

This paper describes a method to produce monodisperse cell-encapsulating microgel beads composed of a self-assembling peptide gel for three-dimensional (3D) cell culture. We used a 3D microfluidic axisymmetric flow-focusing device with an external gelation method. The finely powdered salts were dispersed into a continuous phase, and the salts induced the gelation when in contact with the peptide solution. Over 93% of the cells survived after the encapsulation, and the cells migrated and grew within the gels. Applications of our cell-encapsulating beads include bead-based cell assays in drug testing and engineering tissue constructs.

Monodisperse semi-permeable microcapsules for continuous observation of cells.

We present a method for forming monodisperse semi-permeable microcapsules composed of an alginate-poly-L-lysine (PLL) membrane for the observation of encapsulated cells. These microcapsules were prepared with a monolithic three-dimensional microfluidic axisymmetric flow-focusing device by an internal gelation method using glucono-1,5-lactone in order to provide mild conditions for the cells. The microcapsules were sufficiently monodisperse and robust to be trapped in a bead-based microfluidic array system for easy observation. We also confirmed that (i) the alginate-PLL membrane is semi-permeable so that cells and microorganisms cannot pass through it but nutrients and wastes can, (ii) cells are able to move freely inside the semi-permeable microcapsules, and (iii) cells can be successfully proliferated in the microcapsules.

Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method.

Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100-400 microm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 microm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.

A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization.

The proper function of many tissues depends critically on the structural organization of the cells and matrix of which they are comprised. Therefore, in order to engineer functional tissue equivalents that closely mimic the unique properties of native tissues it is necessary to develop strategies for reproducing the complex, highly organized structure of these tissues. To this end, we sought to develop a simple method for generating cell sheets that have defined ECM/cell organization using microtextured, thermoresponsive polystyrene substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges (50 microm wide, 5 microm deep). Vascular smooth muscle cells cultured on these substrates produced intact sheets consisting of cells that exhibited strong alignment in the direction of the micropattern. These sheets could be readily transferred from patterned substrates to non-patterned substrates without the loss of tissue organization. Ultimately, such sheets will be layered to form larger tissues with defined ECM/cell organization that spans multiple length scales.

Second-generation maskless photolithography device for surface micropatterning and microfluidic channel fabrication.

We have previously reported on a maskless photolithography device for surface micropatterning and microfabrication by modifying a commercially available liquid crystal display projector. For the prototype, 10-microm resolution was achieved by downsizing the image on a 0.7-in. liquid crystal display panel to an area of 8 x 6 mm and projecting it on a fixed stage. Here, we report on a second-generation maskless photolithography device having two novel features. First, the sliding lens system with variable focal distances and exchangeable objective lenses achieves a variable resolution of 2-8 mum. Second, the synchronous control of displayed images generated by a personal computer and the movement of a XY-positioning stage allows for the fabrication of micropatterns over a larger area (over 50 x 50 mm). Here, we show examples fabricated with the two novel features.

Clinical effectiveness of probiotics therapy (BIO-THREE) in patients with ulcerative colitis refractory to conventional therapy.

OBJECTIVE: Intestinal microflora has been implicated in the etiology of ulcerative colitis (UC). Over the past few years, the use of probiotics in UC has gained attention. The aim of this study was to evaluate the efficacy of probiotics therapy for mild to moderate distal UC refractory to conventional therapies. MATERIAL AND METHODS: Twenty patients with mild to moderate distal UC took 9 BIO-THREE tablets per day for 4 weeks. Clinical symptoms and endoscopic findings were evaluated as ulcerative colitis disease activity index (UCDAI) scores before and after administration of BIO-THREE. Fecal samples were collected from all patients before and after probiotics administration, and fecal microflora was analyzed by the terminal restriction fragment length polymorphism (T-RFLP) method. RESULTS: Remission (UCDAI score < or =2) was observed in 45% (9/20) of the patients; response (decrease in UCDAI > or = 3, but final score > or = 3) in 10% (2/20); no response in 40% (8/20); and worsening (UCDAI > 3) in 5% (1/20). T-RFLP analysis indicated that the principal alteration in microflora was an increase in bifidobacteria. CONCLUSIONS: This study showed that administration of BIO-THREE improved the clinical symptoms and endoscopic findings in patients with UC, indicating that administration of BIO-THREE is safe and efficacious for the treatment of UC.

Cellular control of tissue architectures using a three-dimensional tissue fabrication technique.

Tissue engineering seeks to provide regenerated tissue architectures in vitro but has not yet successfully created thick, highly vascularized, multi-functional tissues replicating native structure. We describe a novel method to fabricate pre-vascularized tissue equivalents using multi-layered cultures combining micro-patterned endothelial cells as vascular pre-cursors with fibroblast monolayer sheets as tissue matrix. Stratified tissue equivalents are constructed by alternately layering fibroblast monolayer sheets with patterned endothelial cell sheets harvested from newly developed thermo-responsive micro-patterned surfaces alternating 20 microm-wide cell-adhesive lanes with 60 microm non-adhesive zones. Cell culture substrates covalently grafted with different thermo-responsive polymers permit spatial switching of cell adhesion and detachment using applied small temperature changes. Endothelial cell patterning fidelity was maintained within the multi-layer tissue constructs after assembly, leading to self-organization into microvascular-like networks after 5-day tissue culture. This novel technique holds promise for the study of cell-cell communications and angiogenesis in reconstructed, three-dimensional environments as well as for the fabrication of tissues with complex, multicellular architecture.

Microfluidic devices for size-dependent separation of liver cells.

Liver is composed of various kinds of cells, including hepatic parenchymal cells (hepatocytes) and nonparenchymal cells, and separation of these cells is essential for cellular therapies and pharmacological and metabolic studies. Here, we present microfluidic devices for purely hydrodynamic and size-dependent separation of liver cells, which utilize hydrodynamic filtration. By continuously introducing cell suspension into a microchannel with multiple side-branch channels, cells smaller than a specific size are removed from the mainstream, while large cells are focused onto a sidewall in the microchannel and then separated into two or three groups. Two types of PDMS-glass hybrid microdevices were fabricated, and rat liver cells were successfully separated. Also, cell size, morphology, viability and several cell functions were analyzed, and the separation performances of the microfluidic devices were compared to that of a conventional centrifugal technique. The results showed that the presented microfluidic devices are low-cost and suitable for clinical use, and capable of highly functional separation with relatively high-speed processing.

Adacolumn selective leukocyte adsorption apheresis in patients with active ulcerative colitis: clinical efficacy, effects on plasma IL-8, and expression of Toll-like receptor 2 on granulocytes.

Adacolumn selective granulocyte and monocyte apheresis (GMA) depletes activated leukocytes in patients with ulcerative colitis (UC). However, this per se cannot fully explain the efficacy of GMA. We have investigated the effects of GMA on the expression of toll-like receptors (TLRs) and plasma interleukin-8 (IL-8). Twenty-two patients with clinical activity index (CAI) of 5-17, 15 with total colitis and 7 with left-sided colitis, were included. Each patient could receive up to 10 GMA sessions, at 1 or 2 sessions per week. GMA was added to the patients' ongoing medication following a relapse or worsening UC, but no additional medication was given. Further, at entry and pre-GMA, blood samples were taken for full blood cell count, expression of TLRs on leukocytes, and plasma IL-8. Seventy-five percent of patients achieved remission after the 10th session (CAI, < or =4; P < 0.005) and there was a marked fall in C-reactive protein (P < 0.01), plasma IL-8 (P < 0.001), and granulocytes (P < 0.05) but an increase in lymphocytes (P < 0.05). The expression of TLR2 on granulocytes was down-modulated (P < 0.05) together with suppression of inflammatory cytokines produced by peripheral blood leukocytes. In conclusion, GMA appears to be an effective adjunct therapy to induce remission in the majority of patients, who are then spared from excess drug therapy. The procedure is associated with sustained immunomodulation. Control studies should strengthen these findings.

Heterotypic cell interactions on a dually patterned surface.

It is worth investigating heterotypic cell-cell interactions by mimicking their in vivo structures and environment. In the present study, physiological cellular response and behavior of hepatocytes and endothelial cells were investigated by controlling their contact periphery in a new co-culture system. Rat primary hepatocytes and bovine endothelial cells were co-cultured on a dually patterned surface. Hepatic physiological functions such as albumin secretion and ammonium metabolism were enhanced by increasing heterotypic cell-cell interactions in a patterned co-culture. Furthermore, enhanced hepatic functions through heterotypic interactions are effective within a limited area apart from endothelial cells as evidenced by immunofluorescence staining of hepatic intracellular albumin, indicating that heterotypic interactions act in a paracrine manner. Thus, heterotypic cell communications that play indispensable roles in increasing hepatic physiological functions should be obtained with an increasing periphery of two-cell domains. These findings are important for the reconstruction of complex tissues such as liver and pancreas.

The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets.

Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27 degrees C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm-BMA) co-grafted domains (1-mm laser dot), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37 degrees C, achieving patterned co-cultures. Reducing culture temperature to 20 degrees C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets.

Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces.

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37 degrees C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces.