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Visual field defect is one of the various clinical manifestations in patients with subarachnoid hemorrhage (SAH). Little is known about the pathogenic mechanism of visual field defect in SAH. In the current study, we investigated the diffusion tensor imaging (DTI) finding of the optic radiation in patients with SAH following rupture of a cerebral artery aneurysm. We recruited 21 patients with aneurismal SAH (12 males, 9 females, mean age, 52.67 years; range, 41–68 years) who showed no definite lesion along the visual pathway. Twenty-one age-and sex-matched normal control subjects were also recruited. DTI data were acquired at an average of 5.9 weeks (range: 3–12 weeks) after onset and reconstruction of the optic radiation was performed using DTI-Studio software. The fractional anisotropy value, apparent diffusion coefficient value, and fiber number of the optic radiation were measured. The fractional anisotropy value of the optic radiation was significantly decreased, and the apparent diffusion coefficient value was significantly increased, in patients with aneurismal SAH than in normal control subjects. However, there was no significant difference in the fiber number of the optic radiation between patients with aneurismal SAH and normal control subjects. The decrement of fractional anisotropy value and increment of apparent diffusion coefficient value of the optic radiation in patients with aneurismal SAH suggest optic radiation injury. Therefore, we recommend a thorough evaluation for optic radiation injury in patient with aneurismal SAH.

The corticoreticular pathway (CRP) mainly mediates proximal and axial muscles and therefore it is an important neural tract for walking (Miyai et al., 2002; Matsuyama et al., 2004; Mendoza and Foundas, 2007). Diffusion tensor tractography (DTT), derived from diffusion tensor imaging (DTI), enables reconstruction of the CRP three-dimensionally and several studies have reported on the association between the CRP and gait recovery (Yeo et al., 2012a, 2013; Jang et al., 2013, 2015; Jang and Seo, 2014; Kwon and Jang, 2014). However, to the best of our knowledge, there has been no report on degeneration of the CRP. In the current study, we reported on a patient with intracerebral hemorrhages (ICHs) in both hemispheres, presenting with gait deterioration due to neural degeneration of the CRP as shown on DTT images. A 64-year-old male presented with gait deterioration which started 2 months ago. He had a history of spontaneous ICHs in both putamens (the right ICH: 19 years ago, and the left ICH: 11 years ago). The patient's motor weakness was measured using Medical Research Council (MRC) scale (Council, 1976). His walking ability was assessed using Functional Ambulatory Category (FAC) (Cunha et al., 2002). Before the onset of gait deterioration, his motor function had recovered to a subnormal state in the right extremities and a nearly normal state in the left extremities; therefore, he was able to walk independently, even on stairs (FAC: 4). Since about 2 months ago, his gait function had begun to deteriorate and aggravated progressively with time. Two months after the onset of gait disturbance, he could not walk independently, even on floors (FAC: 1.5). He presented with weakness of proximal joints along with more severe weakness in the right extremities compared with the left side (MRC scale, shoulder abductor: 4-/4, elbow flexor: 4/4+, finger extensor: 4/4+, hip flexor: 4-/4, knee extensor: 4/4+, ankle dorsiflexor: 4/4+) (Council, 1976). Brain MR images taken at 2 months after onset of gait deterioration showed leukomalactic lesions in both subcortical white matter and basal ganglia (Figure 1B). Figure 1 Brain CT and MR images and results of diffusion tensor tractography (DTT) in a 64-year-old male patient with gait deterioration. DTI data were obtained twice (first DTI: 6 years ago and second DTI: 2 months after onset of gait deterioration). Sixty contiguous slices (field of view = 240 × 240 mm2; repetition time = 10,726 ms; echo time = 76 ms; b = 1,000 s/mm2; number of excitations = 1; and thickness = 2.5 mm) were acquired. FACT algorithm was used for fiber tracking. The CRP was reconstructed using fibers passing through two regions of interest (ROIs) on the color map. The first ROI was given at the medullary reticular formation and the second ROI at the midbrain tegmentum. Termination criteria used for fiber tracking were fractional anisotropy (FA) of less than 0.2 and angle of less than 60°. On the first DTT scan performed 6 years ago, the integrities of the CRPs in both hemispheres were preserved from the cerebral cortex to the medulla. By contrast, on the second DTT taken at 2 months after onset of gait deterioration, both CRPs showed discontinuation at the corona radiata level compared with the first DTT (Figure 1C). In this study, the gait deterioration in this patient was ascribed to delayed neural degeneration of both CRPs. The patient began to show gait deterioration 2 months ago, before undergoing the second DTT. The second DTT for the CRP, taken at 2 months after onset of gait deterioration, showed discontinuations at the corona radiata compared with the first DTT (6 years ago), which showed that integrities of both CRPs were intact. This finding was consistent with the characteristics of motor weakness of the patient (more severe weakness of proximal muscles) (Matsuyama et al., 2004; Mendoza and Foundas, 2007; Yeo et al., 2013). Regarding the pathophysiological mechanism of delayed neural degeneration of the CRP in the corona radiata, injury of peri-lesional white matter might occur through a chemical mechanism: a blood clot might release potentially toxic substances which can induce injury to neural tissue, such as free iron, which might release free radicals or inflammatory cytokines (Chua et al., 2009; Yeo et al., 2012b). However, other factors that can deteriorate the gait function such as normal aging or physical deconditioning should be also considered. Accompanying neural degeneration is often observed after stroke (Yu et al., 2009; Puig et al., 2010). Many studies have reported on Wallerian degeneration that started immediately after neuronal injury in the acute stage of stroke (Yu et al., 2009; Puig et al., 2010). Wallerian degeneration is a process characterized by degeneration of axons and their distal part myelin sheath in the central and peripheral nervous system. However, little is known about delayed neural degeneration in stroke patients. To the best of our knowledge, this is the first study to report on neural degeneration of the CRP although a few previous studies have reported on neural degeneration of the CST in stroke patients (Radlinska et al., 2009; Jang ans Seo, 2016). However, DTI might underestimate the neural fibers due to the fiber-crossing effect (Yamada et al., 2009), which should be considered. In conclusion, we report on a patient who showed delayed gait deterioration due to neural degeneration of the CRP. We believe that DTT evaluation of the CRP would be helpful to elucidate the mechanism behind delayed gait deterioration in stroke patients. This work was supported by the National Research Foundation (NRF) of Korea Grant funded by the Korean Government (MSIP), No. 2015R1A2A2A01004073.

A 34-year-old male patient underwent conservative management for traumatic hemorrhage in the right frontal lobe (Figure 1A). The patient lost consciousness for approximately 4 weeks and experienced post-traumatic amnesia continuously from the time of the accident. The patient's Glasgow Coma Scale score (Teasdale et al., 1974) was 6 when he arrived at the hospital. At 5 weeks after onset, he was transferred to the rehabilitation department to undergo rehabilitation. Brain MRI showed malactic lesions in both the frontal lobes and right thalamus (Figure 1B). The patient exhibited impaired consciousness, with a Glasgow Coma Scale score of 9 and Coma Recovery Scale-Revised score of 8 (Giacino et al., 2004). Figure 1 Brain CT images, magnetic resonance images and diffusion tensor tractography (DTT) images of a 34-year-old male patient with traumatic brain injury. Diffusion tensor tractography (DTT) data were obtained at 6 weeks after onset using a 6-channel head coil on a 1.5 T Philips Gyroscan Intera (Philips, Best, the Netherlands) with single-shot echo-planar imaging. Imaging parameters were as follows: acquisition matrix = 96 × 96; reconstructed matrix = 192 × 192; field of view = 240 × 240 mm2; repetition time = 10,726 ms; echo time = 76 ms; parallel imaging reduction factor (SENSE factor) = 2; EPI factor = 49; b = 1,000 s/mm2; number of excitations = 1; and a slice thickness of 2.5 mm. FACT algorithm was used for fiber tracking. For ascending reticular activating system (ARAS) analysis, the seed region of interest (ROI) was given on the pontine reticular formation. According to previous reports (Yeo et al., 2013; Jang et al., 2014), the target ROI was placed on the thalamic intralaminar nuclei to analyze the connectivity of the intralaminar nuclei. The patient showed intact neural connectivity between the thalamic intralaminar nuclei the frontal lobe. However, the left lower ARAS between pontine reticular formation and thalamic intralaminar nuclei was thinner than the right lower one of the same patient and those in five age-matched healthy control subjects (Figure 1C). In this study, we evaluated the ARAS in a patient with severe traumatic brain injury through two portions: First, three-dimensional reconstruction at the lower ARAS between the pontine reticular formation and the thalamic intralaminar nuclei, second, neural connectivity of the upper ARAS between the thalamic intralaminar nuclei and the cerebral cortex. Our results showed that the left lower ARAS was thinner than that of the right side of the patient and those of normal subjects. These findings appear to suggest an injury of the left lower ARAS. This injury was attributed to traumatic axonal injury because the conventional brain MRI of the patient was normal in the left thalamus and brainstem. Before analysis of the ARAS, we assumed that the main lesion would be located in the right ARAS and left upper ARAS because the brain MRI showed malactic lesions in both frontal lobes and the right thalamus. The main lesion site was detected in the left lower ARAS between the thalamic reticular formation and the thalamic intralaminar nuclei (Edlow et al., 2013; Jang et al., 2015a, b). Our results suggest that analysis of the ARAS using DTT would be useful for elucidating the cause of impaired consciousness. However, limitation of DTT with 1.5 T MRI should be considered, because a higher tesla MRI such as 3.0 T, can show better resolution and identify small tract more precisely. This work was supported by the National Research Foundation (NRF) of Korea Grant funded by the Korean Government (MSIP), No. 2015R1A2A2A01004073.

The less affected hemisphere is important in motor recovery in mature brains. However, in terms of motor outcome in immature brains, no study has been reported on the less affected corticospinal tract in hemiplegic pediatric patients. Therefore, we examined the relationship between the condition of the less affected corticospinal tract and motor function in hemiplegic pediatric patients. Forty patients with hemiplegia due to perinatal or prenatal injury (13.7 ± 3.0 months) and 40 age-matched typically developing controls were recruited. These patients were divided into two age-matched groups, the high functioning group (20 patients) and the low functioning group (20 patients) using functional level of hemiplegia scale. Diffusion tensor tractography images showed that compared with the control group, the patient group of the less affected corticospinal tract showed significantly increased fiber number and significantly decreased fractional anisotropy value. Significantly increased fiber number and significantly decreased fractional anisotropy value in the low functioning group were observed than in the high functioning group. These findings suggest that activation of the less affected hemisphere presenting as increased fiber number and decreased fractional anisotropy value is related to poor motor function in pediatric hemiplegic patients.

Diffuse axonal injury (DAI) is axonal and small vessel injury produced by a sudden acceleration of the head by an external force, and is a major cause of death and severe disability (Paterakis et al., 2000). Prognosis is poorer in patients with apparent hemorrhage than in those without (Paterakis et al., 2000). Therefore, it is important to identify the presence and precise position of hemorrhagic foci for a more accurate diagnosis. CT and magnetic resonance imaging (MRI) have long been applied in the diagnosis of DAI, but they are not sensitive enough for the detection of small hemorrhagic foci, and cannot meet the requirements for early diagnosis. A major advance in MRI has been the development of susceptibility weighted imaging (SWI), which has greatly increased the ability to detect small hemorrhagic foci after DAI (Ashwal et al., 2006). In this study, we retrospectively analyzed MRI data for 25 patients with hemorrhagic DAI verified by clinical imaging, and we explored whether SWI was sufficiently sensitive to evaluate hemorrhagic DAI and help accurately assess the severity of the disease.

The ipsilateral motor pathway from the unaffected motor cortex to the affected extremity is one of the motor recovery mechanisms following stroke (Jang, 2011). Because stroke patients who had shown recovery by this mechanism usually showed poorer motor function, compared with patients who showed recovery by other mechanisms, several researchers have considered this mechanism as a maladaptive plasticity (Jang, 2013).

Extensive lesions involving the posterior quadrant of the cerebral hemisphere (temporal, parietal, and occipital lobes) induce intractable epilepsy. These patients are potential candidates for surgical treatment[1]. Maintenance of isolated nerve tissue activity after surgery plays a crucial role in the neuroprotective effects of neurosurgery treatment. Disconnection surgery of the posterior quadrant is used to completely isolate nerve fibers, while blood supply at the isolated lobes is maintained. Subsequently, cavities caused by cystic or necrotic nerve tissues should be reduced as much as possible, to maintain the activity of isolated nerve tissue[1,2,3,4]. In this study, we selected two patients with temporal-parietal-occipital intractable epilepsy, from the Department of Neurosurgery, Tianjin Huanhu Hospital and Tianjin Neurological Institute (Tianjin, China), between February and May in 2012. The two cases underwent post quadrant disconnection. After surgery, activity of temporal-parietal-occipital nerve tissue and the control of epileptic seizures were observed. Case 1: An 11-year-old male, was admitted to Tianjin Huanhu Hospital in February, 2012, and received surgery 1 week later. Postoperative follow-up was performed every 6 months. The medical history showed mild head injury when he was 5 years old, resulting from a scalp laceration without loss of consciousness. The first seizure occurred 1 year later, with the head turning towards the left side, without limb and trunk spasms. He was conscious, and the seizure stopped after 2 minutes. After the second attack, he was diagnosed and treated for epilepsy. Control of seizures failed after 3 anticonvulsant treatments, became refractory, and he still had one seizure per week, while being treated with a combination of 3 anticonvulsants. Preoperative physical examination showed left homonymous hemianopsia. Video-electroencephalography monitoring showed interictal spikes and slow wave complexes in the right central occipital region (Figure 1A1). Magnetic resonance imaging (MRI) of the brain revealed right occipital congenital porencephaly (Figure 1A2). Based upon these findings, this case was diagnosed as simple partial epilepsy. The left hemisphere was not involved during the seizure, because language function was not affected. Video-electroencephalography monitoring and brain imaging results, as well as the clinical features, identified the epileptogenic region as the right post quadrant. A right post quadrant disconnection was therefore performed. Figure 1 Electroencephalogram (EEG) and MRI changes of case 1, an 11-year-old male patient with temporal-parietal-occipital epilepsy, before and after posterior quadrant disconnection. After surgery, the patient orally received 300 mg of oxcarbazepine, twice a day, for an extended period. Follow-up was performed every 6 months. Six month postoperative electroencephalogram showed no epileptic waves (Figure 1B1). MRI revealed complete resection of the right posterior temporal-parietal-occipital lobes, and the majority of isolated brain tissue produced normal signals (Figure 1B2). Case 1 was followed-up for 22 months, during which time no aura or seizure was observed. Case 2: A 28-year-old female was admitted to Tianjin Huanhu Hospital in April, 2012, and received surgery 1 week later. Medical history showed no remarkable features such as brain injury or infection. The first attack occurred 6 months before admission, involving generalized tonic-clonic seizures and disturbance of consciousness, which continued for 1–2 minutes, and then stopped. She was diagnosed with epilepsy and received anticonvulsant medication after a secondary seizure 2 months before admission. However, the seizures were not controlled, and a brain CT scan at a local hospital revealed suspicious “gray matter heterotopia”. Preoperative physical examination showed no seizure attacks during 3 days of video-electroencephalography monitoring. Interictal electroencephalogram showed interrupted short-range low and middle amplitude waves in the right hemisphere. There were also some interictal spikes and slow wave complexes, especially in the temporal and parietal regions (Figure 2A1). MRI of the brain showed an irregular and abnormal signal isodensity with gry matter subendymal at the atrium of the right ventricle. Similar signals were also observed in the temporal horn of the ipsilateral ventricle under the ventricular ependyma (Figure 2A2). The neuroimaging and electroencephalogram therefore showed an epileptogenic focus at the posterior right hemisphere, and posterior quadrant disconnection was performed with the same surgical procedures as case 1. Figure 2 Electroencephalogram (EEG) and MRI changes of case 2, a 28-year-old female patient with temporal-parietal-occipital epilepsy, before and after posterior quadrant disconnection. After surgery, the patient orally received 500 g of sodium valproate, twice a day, for an extended period. A 6 month postoperative electroencephalogram showed no epileptic waves (Figure 2B1). MRI revealed complete resection of the right posterior quadrant, but nerve tissue still survived (Figure 2B2). Case 2 was followed up for 20 months, during which time no seizures were observed. The involved two patients were informed of the experimental scheme and risk prior to treatment, and both patients signed informed consents. Disconnection surgery of the posterior quadrant is used to cut nerve fibers between the posterior quadrant and the resting region in the hemisphere, isolating the epileptogenic zone at the posterior quadrant. In this study, we only partially removed the superior temporal gyrus (tissue width < 5 mm), so that the tissue could be isolated with less incision. A careful macroscopic examination was made to preserve arteries and veins while the incision was made, therefore the isolated lobe maintained a blood supply and survived. MRI scans of the two epileptic patients showed that the disconnected brain tissue still survived, the activity of temporal-parietal occipital nerve tissue was maintained, and nerve fibers were completely disconnected. The involved patients were followed-up for over 1 year after surgery, during which time the epilepsy was completely controlled.

The somatosensory system plays a crucial role in executing precise movements by providing sensory feedback (Farrer et al., 2003; Rabin and Gordon, 2004). Somatosensory dysfunction is a common problem following stroke. In particular, somatosensory impairments, such as impairment in touch, proprioception, light touch, and vibration have been frequently observed (Carey et al., 1993; Sullivan and Hedman, 2008; Tyson et al., 2008). Patients with somatosensory dysfunction show negative effects on motor control, and it sometimes becomes difficult to perform daily activities independently. In addition, these patients require more time to recover functions compared with those without somatosensory deficits (Doyle et al., 2010; Sommerfeld and von Arbin, 2004; Sullivan and Hedman, 2008). Therefore, a better understanding of the recovery mechanism underlying somatosensory dysfunction is necessary for a successful neurorehabilitation outcome.

Ischemic stroke is most commonly caused by vascular occlusion due to thrombosis or arterial embolism. Recently, thrombolysis has been used with increasing frequency for the treatment of acute ischemic stroke. Among the drugs used for thrombolysis, only recombinant tissue plasminogen activator is widely accepted internationally (Albers et al., 2008). In China, urokinase has been widely used for thrombolysis after acute ischemic stroke. Pro-urokinase is the precursor of urokinase. Compared with urokinase, pro-urokinase has greater ability to dissolve thrombus and is safer to use. A previous study found that the recanalization rate was significantly higher after arterial thrombolysis with pro-urokinase than with recombinant tissue plasminogen activator (Fischer et al., 2005). This study compared the thrombolytic effects of pro-urokinase, recombinant tissue plasminogen activator, and urokinase in a dog model of acute cerebral embolism. For each dog used, 10 mL of arterial blood was taken and placed at room temperature for 3 hours to allow natural consolidation. Blood clots were then pressed into a 2-mm diameter cylinder and cut into 2–3 mm lengths. After general anesthesia, dogs were fixed on an operating table and the trachea was intubated. A 5F sheathing canal was placed in the right femoral artery using a modified Seldinger method (Marx et al., 1996). The sheathing canal was advanced into the internal carotid artery guided by digital subtraction angiography. Vascular traveling to the anterior and middle cerebral arteries was observed in the anteroposterior view (Figure ​(Figure1A1A–C). A mixture of autologous blood clots and physiological saline was injected into the internal carotid artery using a 5 mL syringe. If no embolization was observed on digital subtraction angiography after the first injection of blood clots, the process was repeated (Takano et al., 1998; Oureshi et al., 2004; Harris et al., 2007). When anterior or middle cerebral artery embolization was confirmed, the sheathing canal was withdrawn. Digital subtraction angiography was repeated every 30 minutes for 3 hours to confirm embolization (Figure ​(Figure1D1D–F). Figure 1 Digital subtraction angiography images before and after treatment of acute cerebral embolism in dogs using pro-urokinase, recombinant tissue plasminogen activator, or urokinase. Stroke was successfully induced in 24 dogs. These 24 dogs were randomly divided into four groups: (1) Pro-urokinase group: 1.2 × 105 U/kg pro-urokinase was administered via the femoral vein. One-third of the pro-urokinase was dissolved in physiological saline and administered over 3 minutes, and the remainder was dissolved in 100 mL of physiological saline and administered over 30 minutes. (2) Recombinant tissue plasminogen activator group: 1.37 mg/kg of recombinant tissue plasminogen activator was administered via the femoral vein. One-tenth of the recombinant tissue plasminogen activator was administered over 1 minute, and the remainder was administered over 60 minutes. (3) Urokinase group: 2.15 × 106 U/kg urokinase was dissolved in 100 mL of physiological saline and administered by intravenous infusion over 30 minutes. (4) Model group: 100 mL of physiological saline was administered by intravenous infusion over 30 minutes. Digital subtraction angiography was performed every 30 minutes for 3 hours after thrombolysis. Based on assessment of the Thrombolysis In Myocardial Infarction flow grade, the recanalization rate was higher in the urokinase group than in the model group (Table 1, Figure ​Figure1G1G–U). Hematoxylin and eosin staining showed no hematoma in the infarcted area at 3 hours after thrombolysis in any of the groups, but nerve cells in the infarcted tissues showed degeneration, coagulative necrosis, vacuole-like structures, indistinct cell borders, and pyknotic or absent nuclei. In addition, the nerve cells and glial cells were obviously reduced in number or even absent. Infiltration of neutrophilic leukocytes and microglial proliferation or phagocytosis were observed in some regions. There were no obvious differences in cell apoptosis among the groups (Figure ​(Figure2A2A–E). Hemorrhage was observed in the infarcted area in one dog from each of the pro-urokinase and urokinase groups (Figure ​(Figure2F2F–H). Table 1 Effectiveness of pro-urokinase, recombinant tissue plasminogen activator, and urokinase for the treatment of acute cerebral embolism Figure 2 Histological findings in the area of cerebral infarction at 3 hours after thrombolysis (hematoxylin and eosin staining). Previous studies reported that patients who underwent thrombolysis over 3 hours had a high incidence of hemorrhage (Camerlingo et al., 2005). Obvious hematoma was not observed in this dog model of stroke because dogs have abundant collateral cerebrovascular circulation, resulting in a limited area of infarction, and thrombolysis was performed early. The results of this study show that recanalization after thromboembolism was similar after thrombolysis with pro-urokinase and recombinant tissue plasminogen activator, and that both these drugs were more effective than urokinase (both P < 0.05). However, pro-urokinase and recombinant tissue plasminogen activator did not have any definite protective effects against neuronal injury.

The repair of peripheral nerve injuries with autologous nerve remains the gold standard (Wang et al., 2005; Yao et al., 2010; Deal et al., 2012; Kriebel et al., 2014; Liu et al., 2014; Tamaki et al., 2014; Yu et al., 2014; Zhu and Lou, 2014). With advances in tissue engineering and biomaterials, tissue-engineered nerve conduits with various biomaterials and structures, such as collagen and chitosan nerve conduits, have already been used in the clinic as alternatives to autologous nerve in the repair of peripheral nerve injury (Wang et al., 2012; Sviženska et al., 2013; Eppenberger et al., 2014; Gu et al., 2014; Koudehi et al., 2014; Moya-Diaz et al., 2014; Novajra et al., 2014; Okamoto et al., 2014; Shea et al., 2014; Singh et al., 2014; Tamaki et al., 2014; Yu et al., 2014). Therefore, new simple and effective methods are needed to better evaluate the outcomes of repair using nerve conduits in vivo. Ultrasound is a common noninvasive clinical detection modality that has been used in many fields. However, ultrasound has only rarely been used to observe implanted nerve conduits in vivo. Haug et al. (2013) tried to displace the collagen nerve conduit for repairing the digital nerve under ultrasound. Here, we report the first use of ultrasound to noninvasively observe the changes in chitosan nerve conduits implanted in rats over time. Chitosan (Nantong Xincheng Biochemical Company, Nantong, Jiangsu Province, China) was purified twice by dissolution in 10 g of acetic acid, filtration, precipitation with 50 g of NaOH, and finally drying in a vacuum at room temperature. The degree of chitosan deacetylation was 92.3% as measured by titration. After 5 g of chitosan had completely dissolved in 100 mL of 0.15 mol/L hydrochloric acid, 10% gelatin and then 5 g of chitin powder were added while stirring, forming an opaque viscous liquid. The chitin/chitosan mixture was then injected into stainless-steel casting molds, which were then sealed and placed at −12°C for 2–4 hours. The frozen gels were removed and soaked in 4 mol/L NaOH for 4 hours to neutralize any remaining lactic acid and to complete solidification. The conduits were rinsed repeatedly with distilled water to remove any residual NaOH and sodium lactate and lyophilized under a 35–45 mTorr vacuum for 20 hours. The resulting porous conduits were 2 mm inner diameter, 3 mm outer diameter, and 80 mm long (Yang et al., 2011). A total of 21 clean, female, 2-month-old Sprague-Dawley rats were provided by the Experimental Animal Center of Nantong University, China (license No. SCXK (Su) 2008-0010). The animals were housed in a temperature-controlled environment and allowed food and water ad libitum. All experimental protocols were approved by the Administration Committee of Experimental Animals, Jiangsu Province, China, in accordance with the guidelines of the Institutional Animal Care and Use Committee, Nantong University, China. The rats were deeply anesthetized with an intraperitoneal injection of a compound anesthetic (chloral hydrate: 4.25 g, magnesium sulfate: 2.12 g, sodium pentobarbital: 886 mg, ethanol: 14.25 mL, and propylene glycol: 33.8 mL in 100 mL) at a dose of 0.2–0.3 mL/100 g. The skin and muscle were incised to expose the sciatic nerve at the left mid-thigh. An 8-mm segment of the sciatic nerve (from about 10 mm distal to the proximal end to the ischial tuberosity) was resected to produce a 10-mm gap after slight retraction of the distal and proximal stumps. The nerve gap was bridged by a chitosan nerve conduit, and the proximal and distal nerve stumps were inserted into the two ends of the conduit (1 mm was inserted for each end). Then, the muscle layers were closed with sutures, and the skin was closed with wound clips. After surgery, the animals were placed in warmed cages (Yang et al., 2011). At 1, 2, 3, 4, 8, 12, and 24 weeks after surgery (n = 3 in each group), the rats were again deeply anesthetized with the compound anesthetic. A B-mode ultrasound (HIVISION Avius, HITACHI, Chiba Kashiwa, Japan) equipped with a high-resolution linear transducer with a frequency range of 7.5 to 10 MHz and a gel pad serving as an interface between the transducer and fur was used to detect the nerve conduit implanted in the rat. After ultrasound imaging, the surgical site at the left mid-thigh was reopened to expose the nerve conduit. The length and outer diameter were measured with a ruler after photographing the nerve conduit. The ultrasound imaging clearly showed the longitudinal section, as well as the distal and proximal nerves and cross section of the nerve conduit surrounded by muscles (Figure 1). The length and outer diameter of the nerve conduit measured by ultrasound were not different (P > 0.05) than those measured by ruler after dissection at the different time points (Figure 2). Figure 1 Ultrasound imaging of the morphology of a chitosan nerve conduit in a rat model of sciatic nerve defects after implantation Figure 2 Length (A) and outer diameter (B) of the nerve conduit measured by ultrasound and with a ruler after dissection. In addition, decreases in both the length and outer diameter were seen from 12 to 24 weeks. The decrease in length (P < 0.05) from 12 to 24 weeks was more evident, and this difference reflected the degradation mode of the nerve conduit in vivo in rat. There was no evident fracture or collapse of the nerve conduit. However, the two ends of the nerve conduit had clearly shortened at 24 weeks, and a moderate collapse of the cross section was also observed at 24 weeks (Figures ​Figures11 and ​and22). The strain ratio of the nerve conduit was also measured with ultrasound, which reflects the elasticity of the nerve conduit wall. The gradual increase in the strain ratio of the nerve conduit over time suggests that the nerve conduit degraded in vivo (Figure 3). Figure 3 Ultrasound imaging of the elasticity of the chitosan nerve conduit in a rat model of sciatic nerve defects after implantation. Based on these results, the morphological changes of the nerve conduit can be observed by ultrasound imaging in vivo. In addition, the strain ratio measured by ultrasound may be an objective reflection of the degradation of the nerve conduit in vivo. Moreover, any unsatisfactory complications after implantation, such as fracture, collapse, bleeding, or unusual swelling of the nerve conduits, may be easily identified. However, some factors are related to the effect of ultrasound detection closely. A specialized training is necessary to identify the peripheral nerve and nerve conduit for the ultrasound detector. The image resolution is relative to the ultrasound frequency. Different frequencies maybe suitable for different conduits made of different biomaterials. Because the rat was small on volume, which has a limited absorption capacity of biomaterials, the degradation of chitosan in vivo seemed relatively slow. Also we attempted to detect the dog, a bigger animal, with ultrasound and the trend of degradation of conduit was earlier and more evident. Ultrasound, as a noninvasive imaging modality, can be used as a supplementary observation method during conventional animal experiments on peripheral nerve tissue engineering.

Experimental stroke research commonly employs focal cerebral ischemic rat models (Bederson et al., 1986a; Longa et al., 1989). In human patients, ischemic stroke typically results from thrombotic or embolic occlusion of a major cerebral artery, usually the middle cerebral artery (MCA). Experimental focal cerebral ischemia models have been employed to mimic human stroke (Durukan and Tatlisumak, 2007). Rodent models of focal cerebral ischemia that do not require craniotomy have been developed using intraluminal suture occlusion of the MCA (MCA occlusion, MCAO) (Rosamond et al., 2008). Furthermore, mouse MCAO models have been widely used and extended to genetic studies of cell death or recovery mechanisms (Liu and McCullough, 2011). Genetically engineered mouse stroke models are particularly useful for evaluation of ischemic pathophysiology and the design of new prophylactic, neuroprotective, and therapeutic agents and interventions (Armstead et al., 2010). During the past two decades, MCAO surgical techniques have been developed that do not reveal surgical techniques for mouse MCAO model engineering. Therefore, we compared MCAO surgical methods in rats and mice. Forty-five male Sprague-Dawley rats, weighing 240–260 g, and thirty-four male C57BL/6 mice, weighing 20–25 g, were selected for this study. 4-0 and 6-0 monofilament nylon (AILEE Co., Busan, Korea) was used in MCAO surgery. The monofilament was cut into 5-cm (for rats) or 2-cm (for mice) pieces, and the tip of the monofilament was blunted by heating or poly-L-lysine coating (Sigma-Aldrich, St. Louis, MO, USA) (outer diameter of monofilament: 0.4–0.45 mm for rats and 0.15–0.18 mm for mice). All surgical instruments and materials were autoclaved, and the surgical procedure was performed under sterile conditions. This study was approved by the Ethics Committee for Animal Care and Use at Inje University (Approval No. 2012-29), which is certified by the Korean Association of Laboratory Animal Care. The animals were anesthetized with an intraperitoneal injection of Zoletil (tiletamine + zolazepam cocktail at 40 mg/kg or ketamine at 80 mg/kg) and xylazine (10 mg/kg) (Lee et al., 2012). After anesthetic induction, the animals were placed on a heating pad on a surgical table. During the surgical procedure, the body temperature was continuously monitored with a rectal probe and maintained at 36.5–37.0°C. The surgical region was disinfected with povidone-iodine or 70% alcohol. A midline neck incision was made, and the soft tissues over the trachea were gently retracted with a retractor. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated from the vagus nerve. Typically, the CCA bifurcates into the ECA and ICA, which flow toward the cranial and facial regions, respectively, and then the ICA bifurcates into the MCA and pterygopalatine artery (PPA). The occipital artery (OA) originates from the bifurcation point of the ECA which is placed on the side of the ICA (Figure 1A). Two closely spaced permanent knots were then placed at the distal part of the ECA (below the suprathyroid artery) to prevent the backflow of blood. A microvascular clamp was placed in the ICA and transiently proximal to the CCA junction. The tied section of the ECA was dissected using microscissors to insert the monofilament and reach the CCA junction, and a knot was placed below the arteriotomy in the ECA. The microvascular clamp placed in the ICA was removed to allow for filament insertion. The filament was carefully inserted, up to 18 to 20 mm for rats and 9 to 11 mm for mice, into the MCA from the CCA junction (Figure 1A; captured image). After confirmation of MCA blockage, the rat model allowed a blood supply from the CCA, whereas the mouse model allowed a blood supply after the occlusion period. After 60–90 minutes, the filament was carefully withdrawn until the tip was near the arteriotomy. Following removal of the filament, the knot was tightened in the ECA. When reperfusion was confirmed, the neck was sewn using surgical thread. To relieve pain and discomfort in the postoperative period, topical lidocaine gel was applied to the incision region, and the animal received 1.0 mL of normal saline subcutaneously as volume replenishment after the surgery. At 24 hours after the surgery, the animals were sacrificed and analyzed for brain infarction. All procedures had to be finished within 15 minutes, excluding the occlusion and reperfusion time (Figure 1). After 24 hours after reperfusion, infarct volume was calculated using 2% TTC staining method (Figure ​(Figure1C1C–E) (Bederson et al., 1986b; Park et al., 2012). Figure 1 Schematic representation of surgical procedure and quantification of infarct volume in subjected rodents. In summary, to develop standard and high-quality rodent models of stroke, several points should be taken in MCAO: (1) 0.40–0.45 mm outer diameter 4-0 monofilament nylon (for rats) and 0.15–0.18 mm outer diameter of 6-0 monofilament nylon (for mice) by heating or poly-L-lysine coating. (2) Thread insertion length: 18–20 mm (for rats) and 9–11 mm (for mice). (3) Operation period: maximum of 15 minutes. (4) Occlusion period: 60 minutes. (5) MCA occlusion allows CCA reperfusion for rats or bilateral CCA occlusion for mice.

Many studies have examined motor impairments using voxel-based lesion symptom mapping, but few are reported regarding the corresponding relationship between cerebral cortex injury and lower limb motor impairment analyzed using this technique. This study correlated neuronal injury in the cerebral cortex of 16 patients with chronic stroke based on a voxel-based lesion symptom mapping analysis. Neuronal injury in the corona radiata, caudate nucleus and putamen of patients with chronic stroke could predict walking speed. The behavioral measure scores were consistent with motor deficits expected after damage to the cortical motor system due to stroke. These findings suggest that voxel-based lesion symptom mapping may provide a more accurate prognosis of motor recovery from chronic stroke according to neuronal injury in cerebral motor cortex.