A novel animal model of partial optic nerve transection established using an optic nerve quantitative amputator.

BACKGROUND: Research into retinal ganglion cell (RGC) degeneration and neuroprotection after optic nerve injury has received considerable attention and the establishment of simple and effective animal models is of critical importance for future progress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the optic nerves of Wistar rats were semi-transected selectively with a novel optic nerve quantitative amputator. The variation in RGC density was observed with retro-labeled fluorogold at different time points after nerve injury. The densities of surviving RGCs in the experimental eyes at different time points were 1113.69±188.83 RGC/mm² (the survival rate was 63.81% compared with the contralateral eye of the same animal) 1 week post surgery; 748.22±134.75/mm² (46.16% survival rate) 2 weeks post surgery; 505.03±118.67/mm² (30.52% survival rate) 4 weeks post surgery; 436.86±76.36/mm² (24.01% survival rate) 8 weeks post surgery; and 378.20±66.74/mm² (20.30% survival rate) 12 weeks post surgery. Simultaneously, we also measured the axonal distribution of optic nerve fibers; the latency and amplitude of pattern visual evoke potentials (P-VEP); and the variation in pupil diameter response to pupillary light reflex. All of these observations and profiles were consistent with post injury variation characteristics of the optic nerve. These results indicate that we effectively simulated the pathological process of primary and secondary injury after optic nerve injury. CONCLUSIONS/SIGNIFICANCE: The present quantitative transection optic nerve injury model has increased reproducibility, effectiveness and uniformity. This model is an ideal animal model to provide a foundation for researching new treatments for nerve repair after optic nerve and/or central nerve injury.

Microsurgical anatomy of the abducens nerve.

OBJECTIVE: To clarify the oriented classification, relationships, and variations of the abducens nerve and provide a detailed description of its microsurgical anatomic features. METHODS: A microsurgical anatomic dissection of the abducens nerve was performed in 100 specimens obtained from 50 adult cadaveric heads fixed in formalin and two adult cadaveric heads stained with hematoxylin and eosin for histological examination. Important neurovascular and structural relationships of the abducens nerve were observed. RESULTS: The abducens nerve was divided into five segments (cisternal, petroclival, internal carotid artery, fissural, and intraconal). It coursed in the petroclival venous confluence and there was a complex anatomic relationship. Two new types of abducens nerve variations were found. In one type, the duplicated nerve is split into two branches for a limited length in the cavernous sinus (CS). The other is a complex type, which has a complex course and pattern. This type of duplicated abducens nerve has a communicating branch in the cistern and numerous fasciculi in the CS. In addition, the two branches do not accompany each other for the entire course in the CS. CONCLUSION: The vulnerability of the abducens nerve results from diverse factors. The inferolateral trunk, which arises from the intracavernous segment of carotid artery (also called the artery of the inferior CS), is an important landmark for finding the abducens nerve and sympathetic nerve. Variations of the abducens nerve are not rare. Keeping variations of the nerve in mind is important during skull base operations and transvenous endovascular interventions. Understanding the relationship of the abducens nerve with adjacent structures will help us in preparing for safe surgery.

Microsurgical anatomy of the greater superficial petrosal nerve.

OBJECTIVE: To clarify the orientation, classification, and relationships of the greater superficial petrosal nerve (GSPN), and to provide a detailed description on the microsurgical anatomic features and some landmarks to its identification. METHODS: A microsurgical anatomic dissection of the GSPN was studied in 40 specimens obtained from 20 adult cadaveric heads fixed in formalin. The course of the GSPN and its relationship to neighboring anatomic structures were observed. RESULTS: The GSPN could be divided into four segments: intrapetrosal, suprapetrosal, of foramen lacerum, and of pterygoid canal. About 17.5% (7/40) of GSPNs had communication with the glossopharyngeal nerve (GN). According to communication between the GSPN, internal carotid plexus, and GN, the segment of the foramen lacerum could be divided into five types. The middle meningeal artery and internal maxillary artery were the major blood suppliers of the GSPN. The GSPN usually ran parallel with the lesser petrosal nerve, but sometimes they were at angle to each other. CONCLUSIONS: The relationships between the GSPN and its surrounding structures were studied. The vulnerability of the GSPN is attributed to diverse factors. We confirmed the communication branches between the GSPN and the GN. Our study is important to the understanding of the relationship of the GSPN with adjacent structures and will improve further information during skull base operations.

[Related factors of early post-operative prognosis of meningiomas: an analysis of 953 surgical cases].

OBJECTIVE: To study the related factors of early post-operative prognosis of meningiomas. METHODS: The clinical data of 953 patients with meningiomas were recorded and statistically analyzed with χ(2) test of single factor and logistic regression model of multivariate factors. Patient age; tumor size; tumor location; pre-operative complication of patients such as hypertension, diabetes, heart disease and cerebral infarction; the extent of tumor resection; hemorrhagic shock; blood loss or hemorrhagic shock and brain swelling intra-operatively were taken as variables. The prognosis was evaluated by postoperative Karnofsky performance scale. RESULTS: The prognosis was significantly correlated with the patient age, tumor size, tumor location, preoperative cerebral infarction, the extent of tumor resection, blood loss and hemorrhagic shock intra-operatively (P < 0.05). Such factors as tumor size, preoperative cerebral infarction, the extent of tumor resection (Simpson's scale) and intra-operative hemorrhagic shock were independent risk factors of prognosis for meningiomas. Other factors, such as hypertension, diabetes and heart disease, were unrelated with the prognosis of meningiomas (P > 0.05). CONCLUSION: Patient age, tumor location and pre-operative complications of patients maybe affect the early postoperative prognosis of meningiomas. But such factors as tumor size, preoperative cerebral infarction, the extent of tumor resection and intra-operative hemorrhagic shock are independent risk factors for the post-operative prognosis of meningiomas.

Microsurgical anatomy of the ocular motor nerves.

This study was designed to provide anatomic data to help surgeons avoid damage to the ocular motor nerves during intraorbital operations. The microsurgical anatomy of the ocular motor nerves was studied in 50 adult cadaveric heads (100 orbits). Dissections were performed with a microscope. The nerves were exposed and the neural and muscular relationships of each portion of the nerve were examined and measured. The superior division of the oculomotor nerve coursed between the optic nerve and the superior rectus muscle after it left the annular tendon, and its branches entered into the superior rectus muscle and levator muscle. A mean of five fibers (range 3-7) innervated the superior rectus muscle, and a mean of one fiber (range 1-2) followed a medial direction (84%) or went straight through the superior rectus muscle (16%). The inferior division of the oculomotor nerve branched into the medial rectus, inferior rectus and inferior oblique muscles. The trochlear nerve ended on the orbital side of the posterior one-third of the superior oblique muscle in 76 specimens. The abducens nerve ended on the posterior one-third of the lateral rectus muscle in 86 specimens. If the belly of the lateral rectus muscle was divided into three superior-inferior parts, the nerve commonly entered into the middle one-third in 74 specimens. Based on the observed data, microanatomical relationships of the orbital contents were revised.

Denuded human amniotic membrane seeding bone marrow stromal cells as an effective composite matrix stimulates axonal outgrowth of rat neural cortical cells in vitro.

BACKGROUND: Previous studies have shown that axonal outgrowth in the damaged central nervous system is closely related to the local microenvironment. Transplantation of bone marrow stromal cells (BMSC) or BMSC with some biomaterials has been used to treat various central nervous system diseases with some success. In the current study, we investigated if BMSC on denuded human amniotic membrane (DhAM) as a composite matrix could stimulate axonal outgrowth or not. METHOD: After completely removing the cells on the amniotic membrane with a tryptic and mechanical approach, we seeded BMSC on it. The MTS was applied to test the cytotoxicity of DhAM compared with PLGA and PLL. The morphology of the BMSC was observed by light, electronic and laser confocal microscopy. We also used four kinds of substance (PLL, DhAM, BMSC + PLL, BMSC + DhAM) to coculturing with the cortical neurons. Finally, the lengths of axons in each group were studied using the positive axon-specific marker NF-H. FINDINGS: The DhAM was devoid of cellular components and only its intact basement membrane was left. BMSC grew on the substrate and proliferated with a flat to fusiform morphology. In the MTS test, the results indicated that BMSC cultured in DhAM extract had a high survival rate (> 80%). Moreover, the cortical neural axons in the experimental group (BMSC + DhAM) were longer (287.37 +/- 12.72 microm) than in the other groups (P < 0.01). CONCLUSIONS: This study demonstrates that the DhAM was a good carrier to support growth of BMSC and BMSC on DhAM was an effective composite matrix to support the outgrowth of the axons of rat cortical neurons in vitro. Future studies of the use of the composite matrix in disorders are planned.

Specific targeting of gliomas with multifunctional superparamagnetic iron oxide nanoparticle optical and magnetic resonance imaging contrast agents.

AIM: To determine whether glioma cells can be specifically and efficiently targeted by superparamagnetic iron oxide nanoparticle (SPIO)-fluorescein isothiocyanate (FITC)-chlorotoxin (SPIOFC) that is detectable by magnetic resonance imaging (MRI) and optical imaging. METHODS: SPIOFC was synthesized by conjugating SPIO with FITC and chlorotoxin. Glioma cells (human U251-MG and rat C6) were cultured with SPIOFC and SPIOF (SPIO-FITC), respectively. Neural cells were treated with SPIOFC as the control for SPIOFC-targeted glioma cells. The internalization of SPIOFC by glioma cells was assessed by MRI and was quantified using inductively-coupled plasma emission spectroscopy. The optical imaging ability of SPIOFC was evaluated by confocal laser scanning microscopy. RESULTS: Iron per cell of U251 (72.5+/-1.8 pg) and C6 (74.9+/-2.2 pg) cells cultured with SPIOFC were significantly more than those of U251 (6.6+/-1.0 pg) and C6 (7.1+/-0.8 pg) cells incubated with SPIOF. The T2 signal intensity of U251 and C6 cells cultured with SPIOFC (233.6+/-25.9 and 211.4+/-17.2, respectively) were substantially lower than those of U251 and C6 cells incubated with SPIOF (2275.3+/-268.6 and 2342.7+/-222.4, respectively). Moreover, there were significant differences in iron per cell and T2 signal intensity between SPIOFC-treated neural cells (1.3+/-0.3; 2533.6+/-199.2) and SPIOFC-treated glioma cells. SPIOFC internalized by glioma cells exhibited green fluorescence by confocal laser scanning microscopy. CONCLUSION: SPIOFC is suitable for the specific and efficient targeting of glioma cells. MRI and optical imaging in conjunction with SPIOFC can differentiate glioma cells from normal brain tissue cells.

[Time course of calpain activity changes in rat neurons following fluid percussion injury and the interventional effect of mild hypothermia].

OBJECTIVE: To investigate the time course of calpain activity changes in rat neurons following fluid percussion injury (FPI) under normothermia (37 degrees celsius;) and mild hypothermia (32-/+0.5) degrees celsius;. METHODS: In vitro cultured rat neurons were subjected to FPI followed by application of mild hypothermia for intervention at different time points, and the changes in intraneuronal calpain activity following FPI and the interventional effect of mild hypothermia on calpain activity were evaluated by UV-spectrophotometry at different time points. RESULTS: Remarkable changes occurred in calpain activity in the neurons following FPI at 37 degrees celsius;, and mild hypothermia produced obvious interventional effect on calpain activity in close relation to the timing of intervention initiation. CONCLUSION: Intraneuronal calpain activity changes following FPI are involved in the pathological process of cellular injury, and mild hypothermia might offer protection against traumatic brain injury to some extent by regulating calpain activity. The interventional effect of mild hypothermia is associated with the timing of the intervention initiation.

Human neural stem cells promote corticospinal axons regeneration and synapse reformation in injured spinal cord of rats.

BACKGROUND: Axonal regeneration in lesioned mammalian central nervous system is abortive, and this causes permanent disabilities in individuals with spinal cord injuries. This paper studied the action of neural stem cell (NSC) in promoting corticospinal axons regeneration and synapse reformation in rats with injured spinal cord. METHODS: NSCs were isolated from the cortical tissue of spontaneous aborted human fetuses in accordance with the ethical request. The cells were discarded from the NSC culture to acquire NSC-conditioned medium. Sixty adult Wistar rats were randomly divided into four groups (n = 15 in each): NSC graft, NSC medium, graft control and medium control groups. Microsurgical transection of the spinal cord was performed in all the rats at the T11. The NSC graft group received stereotaxic injections of NSCs suspension into both the spinal cord stumps immediately after transection; graft control group received DMEM injection. In NSC medium group, NSC-conditioned medium was administered into the spinal cord every week; NSC culture medium was administered to the medium control group. Hindlimb motor function was assessed using the BBB Locomotor Rating Scale. Regeneration of biotin dextran amine (BDA) labeled corticospinal tract was assessed. Differentiation of NSCs and the expression of synaptophysin at the distal end of the injured spinal cord were observed under a confocal microscope. Group comparisons of behavioral data were analyzed with ANOVA. RESULTS: NSCs transplantation resulted in extensive growth of corticospinal axons and locomotor recovery in adult rats after complete spinal cord transection, the mean BBB scores reached 12.5 in NSC graft group and 2.5 in graft control group (P < 0.05). There was also significant difference in BBB score between the NSC medium (11.7) and medium control groups (3.7, P < 0.05). BDA traces regenerated fibers sprouted across the lesion site and entered the caudal part of the spinal cord. Synaptophysin expression colocalized with BDA positive axons and neurons distal to the injury site. Transplanted cells were found to migrate into the lesion, but not scatter along the route of axon grows. The cells differentiated into astrocytes or oligodendrocytes, but not into the neurons after transplantation. Furthermore, NSC medium administration did not limit the degree of axon sprouting and functional recovery of the injured rats compared to the NSC graft group. CONCLUSIONS: Human embryonic neural stem cells can promote functional corticospinal axons regeneration and synapse reformation in the injured spinal cord of rats. The action is mainly through the nutritional effect of the stem cells on the spinal cord.

[Labeling neural stem cells with superparamagnetic iron oxide in vitro and tracking after implantation with MRI in vivo].

OBJECTIVE: To evaluate the feasibility of monitoring the neural stem cells implanted into the brain by the technique of labeling with superparamagnetic iron oxide (SPIO). METHODS: Neural stem cells were isolated from the cerebral cortex of newborn Wister rats and cultured. SPIO particles and poly-L-lysine were added into the medium to be co-cultured foe one hour. After the formation of neurospheres, Prussian blue staining was conducted and transmission electron microscopy was used to identify the iron particles in these neural stem cells. Sixteen adult female Wistar rats underwent transplantation of labeled neural stem cells into the right side of brain and non-labeled cells were transplanted into the contralateral part as controls. 1, 2, 4, 6, and 7 weeks after the transplantation, MRI examination with the scanning sequences of SE T2WI, FSE T2WI, and GRE T2 * respectively was conducted on the brains of the rats. Four rats at each time point were killed and their brains were taken out to undergo HE staining and Prussian blue staining to track the presence of labeled-cells. RESULTS: After the addition of SPIO the neurospheres continued to proliferate and differentiate normally. Electron microscopy showed vacuolar structures of different sizes under the cytoplasma membrane within and outside which there were high-density iron particles. Prussian blue staining showed numerous blue stained particles in the cytoplasm of the labeled cells. Remarkable low signal change was seen in the right brain transplanted with labeled cells, especially in the condition of scanning sequence of GRET2. Such change could be seen up to 7 weeks after the transplantation. No signal change was found in the left brain. CONCLUSION: SPIO labeling technique is useful in monitoring the outcome of transplanted neural stem cells.

[Effect of intraischemic mild hypothermia on interleukin-1beta and monocyte chemoattractant protein-1 contents in ischemic core of rat cortex after transient focal cerebral ischemia].

OBJECTIVE: To investigate the effect of intraischemic mild hypothermia on the protein levels of interleukin (IL)-1beta and monocyte chemoattractant protein (MCP)-1 in the ischemic core of rat cortex after transient focal cerebralischemia. METHODS: Eighty male Wistar rats were randomly divided into normothermic (37 degrees C) and mild hypothermic (32 - 33 degrees C) groups. The normothermic group was redivided into six subgroups of 8 rats: sham operation, ischemia for 2 hours without reperfusion, and reperfusion for 6 hours, 16 hours, 24 hours, and 48 hours respectively after ischemia; and the mild hypothermic group was redivided into 4 group with 8 rats: reperfusion for 6, 16, 24, and 48 hours. The rats except those in the sham operation subgroup were subjected to right middle cerebral artery occlusion by insertion a specially prepared nylon filament for two hours. Ice bag was used to lower the brain temperature and anal temperature soon after ischemia to 32.0 - 33.0 degrees C within 10 minutes in the mild hypothermic subgroups. The brain and anal temperature remained at 37.0 - 37.5 degrees C in all normothermic subgroups. Then the rats were killed 0, 6, 16, 24 and 48 hours after reperfusion respectively and their brains were taken out to examine the size of brain infarct by 2,3,5-triphenyltetrazolium chloride (TTC) staining reaction. The protein levels of IL-1beta and MCP-1 in the cortical ischemic core were measured by ELISA. RESULTS: No significant change of IL-1beta protein level was found in the cortical ischemic cores at any time point after reperfusion among the normothermic subgroups. The IL-1beta protein levels at different time points were not significantly different between the intraischemic mild hypothermia subgroups and the normothermic subgroups (all P > 0.05). The MCP-1 protein level in the cortical ischemic cores of the normothermic subgroups began to increase since the 6th hour afer reperfusion (22.5 +/- 8.7 ng x g tissue(-1), 17 times that in the sham operation samples, P < 0.05), peaked in 48 hours (110.9 +/- 47.0 ng x g tissue(-1), 83.7 times that in the sham operation sample, P < 0.001). The protein level of MCP-1 in the mild hypothermic subgroups was 8.7 +/- 7.6 ng x g tissue(-1) 6 h after reperfusion (P < 0.005 in comparison with those in sham operation subgroup and ischemia subgroup) and was 56.0 +/- 40.3 ng x g tissue(-1), 48 hours after reperfusion (P < 0.05) incomparison with those in the normothermic subgroups). The sizes of cortical infarct at different time points in the mild hypothermic subgroups were significantly smaller than those in the normothermic subgroups (P < 0.05). CONCLUSION: Mild hypothermia reduces the level of MCP-1 in the cortex after cerebral ischemia/reperfusion which may be one of the important mechanisms of the neuroprotective effects of mild hypothermia.