An ultrasound-guided percutaneous electrical nerve stimulation regimen devised using finite element modeling promotes functional recovery after median nerve transection

Percutaneous electrical nerve stimulation of an injured nerve can promote and accelerate peripheral nerve regeneration and improve function.When performing acupuncture and moxibustion,locating the injured nerve using ultrasound before percutaneous nerve stimulation can help prevent further injury to an already injured nerve.However,stimulation parameters have not been standardized.In this study,we constructed a multi-layer human forearm model using finite element modeling.Taking current density and activated function as optimization indicators,the optimal percutaneous nerve stimulation parameters were established.The optimal parameters were parallel placement located 3 cm apart with the injury site at the midpoint between the needles.To validate the efficacy of this regimen,we performed a randomized controlled trial in 23 patients with median nerve transection who underwent neurorrhaphy.Patients who received conventional rehabilitation combined with percutaneous electrical nerve stimulation experienced greater improvement in sensory function,motor function,and grip strength than those who received conventional rehabilitation combined with transcutaneous electrical nerve stimulation.These findings suggest that the percutaneous electrical nerve stimulation regimen established in this study can improve global median nerve function in patients with median nerve transection.

Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice:effects of 4-aminopyridine

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine(4-AP),a potassium channel blocker,on functional recove ry and muscle atrophy after sciatic nerve crush injury in rodents.However,this effect of 4-AP is unknown in nerve transection,gap,and grafting models.To evaluate and compare the functional recovery,nerve morphology,and muscle atrophy,we used a novel stepwise nerve transection with gluing(STG),as well as 7-mm irreparable nerve gap(G-7/0)and 7-mm isografting in 5-mm gap(G-5/7)models in the absence and presence of 4-AP treatment.Following surgery,sciatic functional index was determined wee kly to evaluate the direct in vivo global motor functional recovery.After 12 weeks,nerves were processed for whole-mount immunofluorescence imaging,and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber crosssectional area and minimal Feret's diameter.Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model.4-AP did not affect the sciatic functional index recovery in any model.Compared to STG,nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting.While muscle weight,cross-sectional area,and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7,4-AP treatment significantly increased right TA muscle mass,cross-sectional area,and minimal Feret's diameter in G-7/0 model.These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap,and 4-AP exerts diffe rential effects on functional recove ry and muscle atrophy.

Serum response factor promotes axon regeneration following spinal cord transection injury

Studies have snown that serum response factor is beneficaial for axonar regeneration of peripheral herves.However,Its role after central nervous system injury remains unclear. In this study,we established a rat model of T9-T10 spinal cord transection injury.We found that the expression of serum response factor in injured spinal cord gray matter neurons gradually increased with time,reached its peak on the 7^(th) day,and then gradually decreased.To investigate the role of serum response factor,we used lentivirus vecto rs to ove rexpress and silence serum response factor in spinal cord tissue.We found that overexpression of serum response factor promoted motor function recovery in rats with spinal cord injury.Qualitative observation of biotinylated dextran amine anterograde tra cing showed that ove rexpression of serum response factor increased nerve fibers in the injured spinal co rd.Additionally,transmission electron microscopy showed that axon and myelin sheath morphology was restored.Silencing serum response factor had the opposite effects of ove rexpression.These findings suggest that serum response factor plays a role in the recovery of motor function after spinal cord injury.The underlying mechanism may be related to the regulation of axonal regeneration.

Etomidate affects the anti-oxidant pathway to protect retinal ganglion cells after optic nerve transection

Our previous studies revealed that etomidate, a non-barbiturate intravenous anesthetic agent, has protective effects on retinal ganglion cells within 7 days after optic nerve transection. Whether this process is related to anti-oxidative stress is not clear. To reveal its mechanism, we established the optic nerve transection injury model by transecting 1 mm behind the left eyeball of adult male Sprague-Dawley rats. The rats received an intraperitoneal injection of etomidate(4 mg/kg) once per day for 7 days. The results showed that etomidate significantly enhanced the number of retinal ganglion cells retrogradely labeled with Fluorogold at 7 days after optic nerve transection. Etomidate also significantly reduced the levels of nitric oxide and malonaldehyde in the retina and increased the level of glutathione at 12 hours after optic nerve transection. Thus, etomidate can protect retinal ganglion cells after optic nerve transection in adult rats by activating an anti-oxidative stress response. The study was approved by the Animal Ethics Committee at Air Force Medical University, China(approval No. 20180305) on March 5, 2018.

Expression of long non-coding RNAs in complete transection spinal cord injury: a transcriptomic analysis

Long non-coding RNAs(lncRNAs)are abundantly expressed in the central nervous system and exert a critical role in gene regulation via multiple biological processes.To uncover the functional significance and molecular mechanisms of lncRNAs in spinal cord injury(SCI),the expression signatures of lncRNAs were profiled using RNA sequencing(RNA-seq)technology in a Sprague-Dawley rat model of the 10th thoracic vertebra complete transection SCI.Results showed that 116 of 14,802 detected lncRNAs were differentially expressed,among which 16—including eight up-regulated(H19,Vof16,Hmox2-ps1,LOC100910973,Ybx1-ps3,Nnat,Gcgr,LOC680254)and eight down-regulated(Rmrp,Terc,Ngrn,Ppp2r2b,Cox6a2,Rpl37a-ps1,LOC360231,Rpph1)—demonstrated fold changes>2 in response to transection SCI.A subset of these RNA-seq results was validated by quantitative real-time PCR.The levels of 821 mRNAs were also significantly altered post-SCI;592 mRNAs were up-regulated and 229 mRNAs were down-regulated by more than 2-fold.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that differentially expressed mRNAs were related to GO biological processes and molecular functions such as injury and inflammation response,wound repair,and apoptosis,and were significantly enriched in 15 KEGG pathways,including cell phagocytosis,tumor necrosis factor alpha pathway,and leukocyte migration.Our results reveal the expression profiles of lncRNAs and mRNAs in the rat spinal cord of a complete transection model,and these differentially expressed lncRNAs and mRNAs represent potential novel targets for SCI treatment.We suggest that lncRNAs may play an important role in the early immuno-inflammatory response after spinal cord injury.This study was approved by the Administration Committee of Experimental Animals,Guangdong Province,China.

Expressions of nestin and glial fibrillary acidic protein in rat retina after optic nerve transection

AIM：To assess the expression of nestin and glial fibrillary acidic protein（GFAP）in rat retina after optic nerve transection.METHODS：Rats were randomly divided into normal control group,sham group and operation group,and used for establishing an animal model of optic nerve transection.Retinal specimen of each group was collected at 3,48h,7and 14d postoperative.Nestin and GFAP expressions on sagittal sections were analyzed by immunohistochemical staining,and protein extraction was analyzed by Western blot.RESULTS：Immunohistochemical analysis showed that nestin positive staining was rarely detected in normal control group and sham group,while sham group showed weak positive staining at 3h postoperative,the reaction gradually increased at 48h postoperative,and reached its maximum at 7d postoperative,and then decreased at 14d postoperative.Compared to the expression of GFAP,there was not statistically significant obvious difference among three groups（P〉0.05）.Result of Western blot method was consistent with that of immunohistochemical method.CONCLUSION：The expression of nestin increased in a time dependent fashion in Müller cells of retina following optic nerve transection,which was statistically significant,but there was no obvious difference in GFAP expression.The results indicate that an increase in colloid synthesis in retina following optic nerve transection can improve the retinal neurons’environment.

Expression of hippocampal corticosteroid receptors,as well as corticotrophin-releasing hormone and vasopressin in the hypothalamic paraventricular nucleus,in fornix transected rats

BACKGROUND： The hippocampus regulates the hypothalamic-pituitary-adrenal axis through negative feedback. The hypothalamic paraventricular nucleus receives neuronal input from the hippocampus via the fomix, OBJECTIVE： To explore whether the negative feedback effect of the hippocampus on the hypothalamic-pituitary-adrenal axis is contributed to the inhibitory effect of mineralocorticoid receptor （MR） and glucocorticoid receptor （GR） in the hippocampus on the paraventricular nucleus via the fornix. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. The study was performed at the Department of Histology and Embryology, China Medical University between September 2006 and September 2008. MATERIALS： Rabbit anti-rat anti-MR and rabbit anti-rat anti-GR antibodies were purchased from Santa Cruz Biotechnology, USA. Rabbit anti-rat anti-corticotrophin releasing hormone （CRH） and rabbit anti-rat anti-arginine vasopressin antibodies were purchased from Wuhan Boster. METHODS： A total of 90 male, Wistar rats were randomly divided into model and sham-surgery groups （n = 45）. Fornix transection was performed in the model group, while the sham-surgery group underwent surgery, but no fornix transection. MAIN OUTCOME MEASURES： Immunohistochemistry was used to examine MR and GR expression in the hippocampus, as well as CRH and anti-arginine vasopressin in the paraventricular nucleus. Western blot was used to measure alterations in MR, GR, and CRH protein expression following fomix transection. RESULTS： Compared with the sham-surgery group, there were no obvious changes in MR and GR expression in the hippocampus, or CRH and anti-arginine vasopressin expression in the paraventdcular nucleus within 4 days of fornix transection. However, after 7-10 days, significantly decreased MR and GR expression in the hippocampus, and increased CRH and anti-arginine vasopmssin expression in the paraventricular nucleus were observed （P 〈 0.05-0.01）. CONCLUSION： Negative feedback from the hippocampus on the hypothalamic-pituitary-adrenal axis might be mediated through the fornix, and the corticosterene actions mediated by hippocampal corticosteroid receptors indirectly modulated the hypothalamic-pituitary-adrenal axis.

Meta analysis of olfactory ensheathing cell transplantation promoting functional recovery of motor nerves in rats with complete spinal cord transection

OBJECTIVE： To evaluate the effects of olfactory ensheathing cell transplantation on functional recovery of rats with complete spinal cord transection. DATA SOURCES： A computer-based online search of Medline （1989-2013）, Embase （1989- 2013）, Cochrane library （1989-2013）, Chinese Biomedical Literature Database （1989-2013）, China National Knowledge Infrastructure （1989-2013）, VIP （1989-2013）, Wanfang databases （1989-2013） and Chinese Clinical Trial Register was conducted to collect randomized controlled trial data regarding olfactory ensheathing cell transplantation for the treatment of complete spinal cord transection in rats. SELECTION CRITERIA： Randomized controlled trials investigating olfactory ensheathing cell transplantation and other transplantation methods for promoting neurological functional recov- ery of rats with complete spinal cord transection were included in the analysis. Meta analysis was conducted using RevMan 4.2.2 software. MAIN OUTCOME MEASURES： Basso, Beattie and Bresnahan scores of rats with complete spinal cord transection were evaluated in this study. RESULTS： Six randomized controlled trials with high quality methodology were included. Meta analysis showed that Basso, Beattie and Bresnahan scores were significantly higher in the olfacto- ry ensheathing cell transplantation group compared with the control group （WMD = 3.16, 95% （21 （1.68, 4.65）; P 〈 0.00001）. CONCLUSION： Experimental studies have shown that olfactory ensheathing cell transplantation can promote the functional recovery of motor nerves in rats with complete spinal cord transection.

The Characteristics of Plant Communities Along East Eurasian Steppe Transect

The East Eurasian Steppe Transect （EEST） is the ifrst international transect across regions of middle and high latitudes in the eastern Eurasian steppe. The EEST is an ideal platform for researching the response of Eurasian temperate steppe to global change, because of its integrated gradients of temperature and human activities on a large-scale. In this study, basic characteristics of plant communities along the EEST across a latitudinal gradient was analyzed. According to the survey of 58 sampling sites, there are 140 species belonging to 34 families and 94 genera. Of particular note was the ifnding of Astragalus dalaiensis which has disappeared in the grasslands of China. On the whole, Gramineae plants are dominant with Liliaceae plants in the communities signiifcantly decreasing along the latitudinal gradient from south to north. The Shannon-Wiener index and biomass of communities all decreased along the latitudinal gradient with signiifcant negative linear regressions. The SDR2 （summed dominance ratio based on two factors） of dominant plants in the upper layers of communities, such as Stipa and Leymus chinensis, decreased along the latitudinal gradient from south to north. Especially, the SDR2 of L. chinensis decreased signiifcantly. The SDR2 of Cleistogenes squarrosa, Agropyron cristatum in the lower layers of communities and the indicator species for degradation were not affected. Potentilla acaulis was found mainly in the southern and northern areas. Stellera chamaejasme was found just in a few sites in the southern area of the EEST. In communities of Stipa grandis and Stipa krylovii, annual and biennial species are dominant. The ratio of annual and biennial species in the community is signiifcantly related to the latitudinal gradient. Perennial herbaceous plants and shrubs were not affected. According to the principal component analysis （PCA）, with the data from 58＆#215;140 dimensions, the ifrst and second components had the lowest proportion, thus indicating that the species compositions and community structures are homogeneous along the EEST. There is a certain degree of spatial differentiation along the EEST due to degradation’s differences resulting from the different land uses.

Normal Alkane Distributions in Soil Samples along a Lhasa-Bharatpur Transect

We present n-alkane distributions in the soil samples along a transect from Lhasa on the Tibetan Plateau to Bharatpur in Nepal, which covers a large geographical area and a wide range of climatic conditions. These data allow us to assess the significance of n-alkane distributions in different vegetation types and their relationships to temperature and precipitation. In the tropical rainforest and broadleaved forest zones, n-alkanes exhibit a bimodal distribution pattern with dominant homologues around n-C_（31） and n-C_（23）. The bimodal distribution of tropical rainforest n-alkanes may be a general pattern because of the presence of many lianas, epiphytic plants, algae and the strong microbial activity and degradation involved in the postdepositional process. In the warm-temperate mixed forest and needle-leaved forest zones, the long-chain alkanes have a pattern of n-C_（31） n-C_（33） n-C_（27）. In the alpine shrub and grassland zone, although the most abundant homologue is n-C_（31） , relatively high n-C_（23） concentrations have also been observed in some samples. The statistical results show a good correlation between n-alkane proxies and climatic factors. The average chain length（ACL） values are positively correlated with precipitation and temperature across the transect. The carbon preference index（CPI） values show a negative correlation with temperature and precipitation along a Lhasa–Bharatpur transect. Although there is a positive relationship between ACL and temperature, it is difficult to separate the two climatic variables（temperature and precipitation） because they are well coupled in the monsoon region.

Variations of plant life form diversity along the Northeast China Transect and its direct gradient analysis

Plant life form diversity and its direct gradient analysis on a larger scale climate change gradient were tested, based on the data from Northeast China Transect platform. The results showed that the species numbers, life form richness and life form diversity were relative higher at the eastern forests and the ecotone between typical vegetation, while those on the meadow grasslands and typical steppes were lower. Although plant life forms can reflect the climate variations, life form diversity is not consistent with the major global gradient along the NECT.

Glial scar size, inhibitor concentration, and growth of regenerating axons after spinal cord transection

A mathematical model has been formulated in accordance with cell chemotaxis and relevant experimental data. A three-dimensional lattice Boltzmann method was used for numerical simulation. The present study observed the effects of glial scar size and inhibitor concentration on regenerative axonal growth following spinal cord transection. The simulation test comprised two parts： （1） when release rates of growth inhibitor and promoter were constant, the effects of glial scar size on axonal growth rate were analyzed, and concentrations of inhibitor and promoters located at the moving growth cones were recorded. （2） When the glial scar size was constant, the effects of inhibitor and promoter release rates on axonal growth rate were analyzed, and inhibitor and promoter concentrations at the moving growth cones were recorded. Results demonstrated that （1） a larger glial scar and a higher release rate of inhibitor resulted in a reduced axonal growth rate. （2） The axonal growth rate depended on the ratio of inhibitor to promoter concentrations at the growth cones. When the average ratio was 〈 1.5, regenerating axons were able to grow and successfully contact target cells.

Effects of targeted muscle reinnervation on spinal cord motor neurons in rats following tibial nerve transection

Targeted muscle reinnervation(TMR)is a surgical procedure used to transfer residual peripheral nerves from amputated limbs to targeted muscles,which allows the target muscles to become sources of motor control information for function reconstruction.However,the effect of TMR on injured motor neurons is still unclear.In this study,we aimed to explore the effect of hind limb TMR surgery on injured motor neurons in the spinal cord of rats after tibial nerve transection.We found that the reduction in hind limb motor function and atrophy in mice caused by tibial nerve transection improved after TMR.TMR enhanced nerve regeneration by increasing the number of axons and myelin sheath thickness in the tibial nerve,increasing the number of anterior horn motor neurons,and increasing the number of choline acetyltransferase-positive cells and immunofluorescence intensity of synaptophysin in rat spinal cord.Our findings suggest that TMR may enable the reconnection of residual nerve fibers to target muscles,thus restoring hind limb motor function on the injured side.

Identification of Differentially Expressed Proteins and Validation of the Changes of N-Ethylmaleimide-sensitive Factor in Rats with Focal Cerebral Ischemia after Transection of the Cervical Sympathetic Trunk

Stellate ganglion blockade （SGB） protects patients from focal cerebral ischemic injury, and transection of the cervical sympathetic trunk （TCST） in a rat model can mimic SGB in humans. The purpose of this study was to investigate the mechanisms underlying the neuroprotective effects of TCST on neuronal damage in the hippocampus in a rat model of middle cerebral artery occlusion （MCAO） in an attempt to elucidate the neuroprotective effects of SGB. The modified method of Zea Longa was used to establish the permanent MCAO model. Male Wistar rats were randomly divided into three groups： sham-operated group, MCAO group, and TCST group. The animals in TCST group were sacri- ficed 48 h after TCST which was performed after the establishment of the MCAO model. Proteins were extracted from the ipsilateral hippocampus and analyzed by two-dimensional difference gel electropho- resis （2D-DIGE） and peptide mass fingerprinting （PMF）. The levels of N-ethylmaleimide-sensitive fac- tor （NSF） were measured as well. The results showed that 11 types of proteins were identified by 2D- DIGE. The expressions of eight proteins were changed both in the sham-operated and TCST groups, and the expressions of the other three proteins were changed in all three groups. Moreover, the expres- sion of NSF was higher in the TCST group than in the MCAO group but lower in the MCAO group than in sham-operated group. The ratio of NSF expression between the MCAO group and sham- operated group was -1.37 （P〈0.05）, whereas that between the TCST group and MCAO group was 1.35 （P〈0.05）. Our results imply that TCST increases the expression of NSF in the hippocampus of adult rats with focal cerebral ischemia, which may contribute to the protection of the injured brain. Our study pro- vides a theoretical basis for the therapeutic application of SGB to patients with permanent cerebral ischemia.

Dark rearing maintains tyrosine hydroxylase expression in retinal amacrine cells following optic nerve transection

The present study examined changes in retinal tyrosine hydroxylase （TH） expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5 7 days. The number of TH-positive synaptic particles correlated with the TH levels indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage （5 7 days after optic nerve injury） in retinal amacrine cells.

A floristic survey of the Hyrcanian forests in Northern Iran, using two lowland-mountain transects

We investigated the floristic composition of the Hyrcanian forests and the related forest-steppe ecotone in Northern Iran by using two long ecological transects, from lowland to upper mountain areas. The study was conducted during 2008 and 2009 and yielded the identification of 395 plant taxa belonging to 233 genera and 78 families. Dicots with 300 taxa were the richest groups of the flora, followed by monocots with 75 taxa, pteridophytes with 18 species,and gymnosperms with two species. The largest families were Asteraceae（33 taxa）; Rosaceae（32 taxa）; and Poaceae（30 taxa）, and the most diverse genera included Carex（15 taxa）; Alchemilla（7 taxa）; and Poa, Geranium and Acer（6 taxa each）. Hemicryptophytes were the most dominant life forms in the area（40 %）; followed by geophytes（31.4 %）; phanerophytes（15.4 %）; therophytes（11.4 %）; and chamaephytes（1.8 %）. Phytogeographically,Euro-Siberian/Irano-Turanian elements（86 taxa, 21.8 %）and Euro-Siberian elements（85 taxa, 21.5 %） were the most common chorotypes in the area. Out of 395 taxa, 66taxa（16.7 %） were endemics and subendemics in Iran, of which 26 taxa were exclusively endemics of Iran.According to the IUCN Red List Categories, 48 threatened plant taxa were found in the study area. Plant diversity, life form, and chorotypes in the current study were compared with similar transect studies in other areas of the Hyrcanian forests and in different altitudinal belts, using S？renson similarity indices. Floristic composition of the surveyed transects demonstrated almost 50 % similarity between them.

The Geoscience Transect from Xiangshui,Jiangsu Province to Mandal,Inner Mongolia

The Xiangshui to Mandal geoscience transect, which has a total length of 1,200 km, extends diagonally,from SE to NW, across the Sino-Korean plate and its ancient continental margin, passing through nine tectonicunits: the Subei-Jiaonan terrane, West Shandong block, North China rift basin, Taihang-Wutai block, Ordosblock, Hu-Bao (Hohhot-Baotou) Basin, Yinshan block and Inner Mongolian fold system. The graphic display of the transect is compiled according to the Guidelines for the Global GeoscienceTransect (GGT) Project on the basis of an integrated study of all available geological, geochemical andgeophysical data, thus bringing light to the process of cratonization of the Sino-Korean plate and its relation-ship with the transitional zone of the ancient continental margin, and the features of intraplate structures pro-duced by subsequent tectonic modifications and intraplate dynamics.

The Yadong-Golmud Geoscience Transect in the Qinghai-Tibet Plateau

On the basis of abundant geological and geophysical data. 6 terranes have been distinguished on theQinghai-Tibet Plateau. The plateau is a single integrated lithospheric unit although it is divided into blocks.With Amdo as a boundary. the crust may be divided into two parts with different crustal structures. The struc-ture in the southern part is complex, while that in the northern part is simple. The current study has revealedthat 8 factors such as slab subduction. overthrust and superimposition are responsible for crustal shorteningand thickening in the region. The uplift of this region is possibly due to northward compression of the Indianplate and southward compression of the Eurasian plate with the former predominating. The compression led tothe asthenospheric movements which were also influenced by thermal activity caused by doming at the bound-ary between the core and mantle.

Laparoscopic step 1 ALPPS with microwave transection of theliver for Klatskin tumors:Is it worthwhile?

To the Editor：Associating liver partition and portal vein ligation for stagedhepatectomy （ALPPS） was introduced in 2007 [1]. This techniqueis now used worldwide and has a wide spectrum of indications.However, scientific debate and un-regulated surgical experimentationconcerning the application of this technique led the surgeons to organize the first Consensus Meeting [2] to build a scientificconsensus. One of the most controversial indications to ALPPS isits application for Klatskin tumors. The aim of this letter is to analyzethe critical aspects of laparoscopic microwave ALPPS for radicalsurgical treatment of Klatskin tumors.

Inhibition of ciliary neurotrophic factor in a rat model of transected spinal cord

Ciliary neurotrophic factor （CNTF） dramatically increases following spinal cord injury and participates in the repair process, although some studies have shown that CNTF plays a role in promoting glial scar formation following spinal cord injury. The antibody closure model can be used to inhibit CNTF expression following spinal cord injury, thereby furthering the understanding of the role of CNTF in spinal cord injury repair. In the present experiment, spinal catheters were placed in the vertebral canal of spinal cord transected rats, and CNTF antibodies were injected following fixation of the paraspinal muscle catheter. At 24 hours after a single CNTF antibody injection, CNTF expression decreased in the thoracic and lumbar spinal cord and recovered to normal levels by 48 72 hours. CNTF antibody treatment can effectively block endogenous CNTF expression in the thoracic and lumbar spinal cord during an interval of less than 24 hours in transected rats.