Oxygen-Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats.

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.

Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co2+) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co2+ for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co2+ induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co2+ exposure.

Possible utility of contrast-enhanced ultrasonography for detecting spread of local anesthetic in nerve block.

PURPOSE: This study demonstrated the effects of perfluorobutane (Sonazoid®) with contrast-enhanced ultrasonography (CEUS) to identify the spread of local anesthetics in ultrasound-guided nerve block. METHODS: This study consists of simulation, cadaveric, and animal studies. In a simulation study, 1% lidocaine with 10- to 1000-fold diluted Sonazoid®, a US-specific contrast agent to diagnose hepatic and breast cancers (0.5 mL), was injected into a resin-based phantom to determine the optimal concentration for ultrasound-guided peripheral nerve block. The enhanced area was measured by direct observation and ultrasonography (US). In the cadaver study, ultrasound-guided sciatic nerve block was performed at the popliteal fossa in the 9 extremities, and 5 mL of the optimally diluted Sonazoid® defined in the simulation study with X-ray contrast medium and blue dye was injected. Longitudinal spread of the solution was measured by CEUS, X-ray imaging and anatomical dissection. In the animal study, the optimally diluted Sonazoid® was injected around the sciatic nerve of rats (n = 6), and neuronal function and toxicity were evaluated by behavioral and histological estimation. RESULTS: The simulation study proved that 100-fold diluted Sonazoid® was the optimal concentration. In the cadaver study, CEUS and anatomical dissection (r = 0.90, P = 0.0020) or radiography (r = 0.84, P = 0.0072) showed high agreement and correlation with the longitudinal spread. CEUS clearly showed a fine intraneuronal injection image compared to the usual B-mode imaging. The animal study suggested no adverse effects by co-administration of lidocaine and Sonazoid®. CONCLUSIONS: CEUS with 100-fold diluted Sonazoid® could identify the spread of local anesthetic as well as radiography and anatomical dissection, and distinguish between intra- and extraneuronal injections without neurodegeneration.

Integrated care through disease-oriented clinical care pathways: experience from Japan's regional health planning initiatives.

INTRODUCTION: In April 2008, Japan launched a radical reform in regional health planning that emphasized the development of disease-oriented clinical care pathways. These 'inter-provider critical paths' have sought to ensure effective integration of various providers ranging among primary care practitioners, acute care hospitals, rehabilitation hospitals, long-term care facilities and home care. DESCRIPTION OF POLICY PRACTICE: All 47 prefectures in Japan developed their Regional Health Plans pursuant to the guideline requiring that these should include at least four diseases: diabetes, acute myocardial infarction, cerebrovascular accident and cancer. To illustrate the care pathways developed, this paper describes the guideline referring to strokes and provides examples of the new Regional Health Plans as well as examples of disease-oriented inter-provider clinical paths. In particular, the paper examines the development of information sharing through electronic health records (EHR) to enhance effective integration among providers is discussed. DISCUSSION AND CONCLUSION: Japan's reform in 2008 is unique in that the concept of 'disease-oriented regional inter-provider critical paths' was adopted as a national policy and all 47 prefectures developed their Regional Health Plans simultaneously. How much the new regional health planning policy has improved the quality and outcome of care remains to be seen and will be evaluated in 2013 after the five-year planned period of implementation has concluded. Whilst electronic health records appear to be a useful tool in supporting care integration they do not guarantee success in the application of an inter-provider critical path.

Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in cerebellar Purkinje cells.

In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.

[Draft of guidelines for human body dissection for clinical anatomy education and research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

[Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

Tricellulin is expressed in autotypic tight junctions of peripheral myelinating Schwann cells.

Autotypic tight junctions are formed by tight junction-like structures in three regions of myelinating Schwann cells, the paranodal loops, Schmidt-Lanterman incisures, and outer/inner mesaxons, and various tight junction molecules, including claudin-19 and junctional adhesion molecule (JAM)-C. Our findings demonstrate the identification and subcellular distribution of a novel tricellular tight junction protein, tricellulin (TRIC), in the autotypic tight junctions of mouse myelinating Schwann cells, compared with the autotypic adherens junction protein E-cadherin and the autotypic tight junction protein JAM-C, which are expressed in the paranodal loops, Schmidt-Lanterman incisures, and mesaxons. In real-time RT-PCR, the expression level of TRIC mRNA was about 10-fold higher in the sciatic nerve than in the spinal cord or cerebrum. In immunostaining, TRIC signals were completely restricted to the peripheral nervous system (PNS) and strongly concentrated at the paranodal loops, Schmidt-Lanterman incisures, and mesaxons of myelinating Schwann cells. In addition, TRIC was expressed in the thin region of the paranode and there was a gap between TRIC and the Na+ channel. Furthermore, TRIC was more distally located from the node than E-cadherin and was colocalized with JAM-C. It is possible that TRIC may be a component to maintain the integrity for PNS myelin function and morphology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The acid-sensing ion channel 2 (ASIC2) of ciliated cells in the developing rat nasal septum.

The airway epithelium is exposed to an acidic environment in certain conditions. The acid-sensing ion channel 2 (ASIC2) belongs to the epithelial amiloride-sensitive sodium channel and degenerin (ENaC/DEG) family and is expressed on cilia of the respiratory epithelium. The aim of this study was to detect the expression of ASIC2 in the nasal septum in the embryonic stage of the rat. ASIC2 expression was not observed in the primary cilium but was found in some cilia on embryonic day 17 (E17). After E18, all cilia showed ASIC2 immunoreactivity. RT-PCR analysis revealed that ASIC2b, a subtype of ASIC2, was expressed in the nasal septum while ASIC2a was not. Quantitative Real-time RT-PCR studies indicated that the expression level of ASIC2 mRNA was highest on E21, just before birth. These results imply that ASIC2 plays little part in the development of the nasal septum epithelium. On the other hand, ASIC2, especially ASIC2b, may function for the survival and retention of ciliated cells of the nasal septum against dynamic changes in the pH environment at birth.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth.

BACKGROUND: Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. METHODS: DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. RESULTS: DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8-18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8-50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. CONCLUSION: Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.

Histotopographical study of human periocular elastic fibers using aldehyde-fuchsin staining with special reference to the sleeve and pulley system for extraocular rectus muscles.

The aim of this study was to investigate the detailed configuration of periocular elastic fibers. Semiserial paraffin sections were made using 40 whole orbital contents from 27 elderly cadavers and stained by the aldehyde-fuchsin method. Periocular tissues were classified into three types according to directions of the elastic fibers, i.e., tissues containing anteroposteriorly running elastic fibers, those with mediolateral fibers, and those with meshwork of fibers. Anteroposterior elastic fiber-dominant tissue was seen in the upper eyelid and newly defined pulley plate for the medial and lateral recti (MR, LR). Mediolateral fibers were predominant in the central part of the inferior rectus pulley. In the pulley plates for the MR and LR, anteroposteriorly running fibers encased the striated muscle. Tenon's capsule and the epimysium of the recti were mediolateral fiber-dominant. However, at the entrance of the muscle terminal where Tenon's capsule reflects and continues to the epimysium, composite elastic fibers provided a meshwork-like skeleton. The elastic mesh was also seen around the lacrimal canaliculi. The pulley for the recti seemed to be composed of two parts--a connective tissue plate encasing the recti and specialized Tenon's capsule at an entrance or porta of the muscle. For both parts, elastic fibers were major functional components. The anteroposterior elastic fibers in the MR and LR pulley plates, especially, seemed to receive anteroposteriorly directed stress and tension from these striated muscles. The elastic interfaces seemed to prevent any concentration of stress that would interfere with periocular striated muscle functions, including hypothetical active pulleys.

Understanding anatomy of "hilus" of detrusor nerves to avoid bladder dysfunction after pelvic surgery: demonstration using fetal and adult cadavers.

OBJECTIVES: To examine the anatomy of the autonomic nerves entering the bladder using cadavers. After pelvic surgery other than cystectomy, urologists and gynecologists have great concern regarding dysuria and urinary retention postoperatively. METHODS: We applied histologic observation of fetuses to findings in adults, because the topographic anatomy of nerves tends to be easier to understand in fetuses than in adults. We examined 5 fetal (2 males and 3 females) and 8 adult (5 males and 3 females) cadavers using histologic sections with hematoxylin-eosin staining and tyrosine hydroxylase immunostaining. RESULTS: In the female adults, as well as in the male and female fetuses, the bladder detrusor nerves were concentrated in a limited area of the superodorsal surface of the bladder at the superomedial aspect of the ureterovesical junction (ie, the "nerve hilus"). Notably, in female adults, these nerves were loosely fasciculated in a fascial pedicle. However, the hilus was not as evident in male adults in contrast to the dense innervation of the seminal vesicles. The bladder detrusor nerves were characterized by a high concentration of tyrosine hydroxylase immunoreactive-negative fibers in contrast to the nerves to the seminal vesicles, which showed immunoreactive-positive fibers. CONCLUSIONS: Detrusor nerves converged at a small area between the bladder and juxtavesical ureter, and these nerves enter the bladder at the "hilus." Detrusor nerve damage near the ureterovesical junction during pelvic surgery should receive more attention to avoid postoperative bladder dysfunction.

Influence of elbow flexion angle on mobilization of the proximal radio-ulnar joint: a motion analysis using cadaver specimens.

The purpose of this study was to determine the most effective elbow joint flexion angle for mobilization of the proximal radio-ulnar joint. Five fresh-frozen cadaveric elbows were used to measure displacement of the radial head in the antero-medial and postero-lateral directions by traction force of 2kgf and 4kgf, respectively. Simulation of the gliding of the proximal radio-ulnar joint was performed at four elbow flexion angles (0 degrees , 30 degrees , 60 degrees , 90 degrees). Data obtained from those flexion angles were compared using one-way repeated measures analysis of variance. Radial head displacement at 60 degrees and 90 degrees during antero-medial gliding were significantly greater than those at 0 degrees and 30 degrees (p<0.05) There were no significant differences in radial head displacement among four elbow flexion angles during postero-lateral gliding at 2kgf and 4kgf. Our findings suggest that proximal radio-ulnar joint mobilization in the antero-medial direction can be performed effectively at 60 degrees and 90 degrees elbow flexion.

Histotopography of the female cavernous nerve: a study using donated fetuses and adult cadavers.

The purpose of this study was to identify the histotopography of the female cavernous nerve. The study used semi-serial horizontal sections of seven fetuses and ten adult cadavers. In fetuses, the female cavernous nerve ran anteriorly between the distal vagina and the levator ani and entered the corpus cavernosum clitoridis. Its course through the argental hiatus was similar to that of the male cavernous nerve. However, the fascial arrangement along the female cavernous nerve was different from that of the three male fetuses included in this study. In female adults, the putative cavernous nerve was found running along the lateral aspect of the rhabdosphincter, along the superior surface of the urethrovaginal sphincter and around the lateral end of the latter sphincter. Because the female cavernous nerve and lateral vaginal wall are closely related, surgical treatment along the mid- and distal urethra should be conducted cautiously so as not to injure the perivaginal autonomic nerves.

The range of glenohumeral joint motion in activities of daily living after rotator cuff repair: a cadaveric biomechanical study.

This study was conducted to quantify the restriction of glenohumeral motion and activities of daily living (ADL) after rotator cuff repair. Eight fresh cadaveric shoulders were used. The ranges of passive shoulder motions in models of the intact cuff tendon, and repaired small cuff and large cuff tears were measured using a 6-degree-of-freedom electromagnetic tracking device. The range of motion for reach, perineal care, hair combing, and washing the axilla were also measured as simulated ADL. Elevation, horizontal adduction, and abduction were significantly restricted in the repaired small-tear model, whereas glenohumeral motion in all directions was significantly restricted in the repaired large-tear model. The repair of the small tear significantly restricted perineal care alone, whereas the repair of the large tear significantly restricted all ADL except hair combing. These findings could be helpful in managing restricted shoulder motion and in instructing patients in acceptable ADL after rotator cuff repair.

Fetal development of the human gubernaculum with special reference to the fasciae and muscles around it.

Previous descriptions of human gubernacular embryology failed to follow some basic developmental processes, and surgically relevant structures, such as the iliopubic tract, had not been discussed relative to gubernacular development. We addressed these shortcomings in this study that examined two stage-groups of human fetuses. At 8-12 weeks of gestation, the gubernaculum arose from the mesonephric fold at or near the gonad. Gubernacular mesenchyme communicated with the subcutaneous tissue via a narrow slit in the rectus aponeurosis. The inguinal fold, containing the inferior epigastric vessels, was separated from the gubernaculum. At 20-25 weeks of gestation, the gubernaculum connected to the testis or uterus. When the testis successfully descended to a peritoneal recess on the lateral side of the umbilical artery, the gubernaculum connected to the testis free of interference by the thick artery and its associated peritoneal fold. This may explain the known asymmetry in testicular descent. The inguinal canal was enclosed by a sheet-like aponeurosis: its ventromedial part was composed of the rectus sheath and the external oblique aponeurosis, whereas the dorsolateral part consisted of a thick aponeurosis covering or facing the iliopsoas. The former (latter) aponeurosis seemed to develop into the inguinal ligament (the iliopubic tract) in adults. According to the topohistology of the muscles associated with the interfoveolar ligament, we identified muscle fragments around the gubernaculum as derivatives of the transversus and/or internal oblique. Consequently, the inguinal canal contained the cremaster proper developing within the gubernaculum and parts of the abdominal wall muscles mechanically incorporated into the canal.

Expression of ASIC2 in ciliated cells and stereociliated cells.

Acid-sensing ion channel 2 (ASIC2) plays a role as a mechanorecptor and acid receptor in the peripheral and central nervous systems. However, several recent studies have suggested that ASIC2 is expressed in several organs, in addition to the nervous system. We have examined the expression and distribution of ASIC2 in rat ciliated cells (trachea and oviduct) and stereociliated cells (epididymis, Corti organ, and ampullary crest) by immunohistochemistry and transmission electron microscopy (TEM). Immunohistochemistry revealed that ASIC2 was expressed in both ciliated cells and stereociliated cells, but the localization differed between these cell types. In ciliated cells, ASIC2 was coexpressed with a cilial marker (acetylated tubulin). In stereociliated cells stained with a stereocilial marker (phalloidin), ASIC2 was observed in the cell body. Observation by TEM suggested that ASIC2 expression was present at the apical side of the cilial membrane in ciliated cells and at the apical side of the cell body in stereociliated cells. This study thus indicates that the proton receptor ASIC2 is expressed in both ciliated and stereociliated cells.

Development of the human hypogastric nerve sheath with special reference to the topohistology between the nerve sheath and other prevertebral fascial structures.

Semi-serial sections from the lumbosacral region of nine fetuses (8-25 weeks gestation) were examined to clarify the lumbar prevertebral fascial arrangement. The prevertebral fasciae became evident after 12 weeks of age. After 20 weeks of age, the hypogastric nerve (HGN) was sandwiched by two fascial structures; the ventral fascia which seemed to correspond to the mesorectal fascia, whereas the dorsal fascia corresponded to the presacral fascia. These fasciae or the HGN sheaths extended laterally along the ventral aspects of the great vessels and associated lymph follicles. The ventral fascia is, to some extent, fused with the mesocolon descendens on the left side of the body. Notably, the lateral continuation of these two fasciae also sandwiches the left ureter, but not the right ureter, presumably due to modifications by the left-sided fusion fascia. A hypothetical common sheath for the HGN and ureter (i.e., the ureterohypogastric or vesicohypogastric fascia) might thus be an oversimplification. Before retroperitoneal fixation, the morphology of the peritoneal recess along the mesocolon descendens and mesosigmoid suggested interindividual differences in location, shape, and size. Therefore, in adults the ease of surgical separation of the rectum and left-sided colon from the HGN seems to depend on interindividual differences in the development of the embryonic peritoneal recess. On the caudal side of the second sacral segment, fascial structures were restricted along and around the HGN, pelvic splanchnic nerve, and pelvic plexus. The rectal lateral ligament thus seems to represent a kind of migration fascia formed by mechanical stress.

Strain reduction of the extensor carpi radialis brevis tendon proximal origin following the application of a forearm support band.

STUDY DESIGN: Experimental laboratory design. OBJECTIVES: To measure the strain at the proximal origin of the extensor carpi radialis brevis (ECRB), and to determine the influence of a forearm support band. BACKGROUND: A forearm support band is often used with the intent to decrease stresses around the origin of the wrist extensors. However, the influence of the location of the band has not been studied. METHODS AND MEASURES: The forearm support band was applied on 8 cadaver arms (mean +/- SD age, 78.4 +/- 10.3 years) and 2 experimental conditions were performed. First, strain measurements were made without applying tension to the distal ECRB tendon, then strain measurements were made with a traction force of 21.5 N being applied to the distal ECRB tendon. Strain of the proximal origin of the ECRB, 1.0 cm distal from the lateral epicondyle, was recorded using a strain gauge. The band was mounted on the forearm at distances equal to 80%, 70%, 60%, 50%, 40%, 30%, and 20% of the forearm length as measured from the wrist. Testing order was randomized. Tension applied to the band was 19.6 N. RESULTS: When no tension was applied to the ECRB, there was no statistically significant difference (P>.05) in strain values at the ECRB origin by mounting the band at any of the forearm positions. In the tension condition, the average (SD) strain with no band was 2.40% (1.40%). The average strain value of 0.85% (0.65%), when the band was mounted 80% of the forearm length proximal to the wrist, was statistically smaller than that obtained without the band (P<.05). CONCLUSIONS: The strain on the ECRB origin was less when the forearm support band was applied 80% proximal from the wrist joint. LEVEL OF EVIDENCE: Therapy, level 5.