We thank Ian Bolding for assistance with surgical preparation of subjects and Elizabeth Sumner for her meticulous editing. These studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research.

<ol>
	<li>Sell, S. L., Johnson, K., DeWitt, D. S., Prough, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+Behavioral+Deficits+in+Rats+after+Fluid+Percussion+Injury.">Persistent Behavioral Deficits in Rats after Fluid Percussion Injury.</a> J Neurotrauma. 34 (5), 1086-1096 (2017).</li><li>Gurdjian, E. S., Lissner, H. R., Webster, J. E., Latimer, F. R., Haddad, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+experimental+concussion:+relation+of+physiologic+effect+to+time+duration+of+intracranial+pressure+increase+at+impact.">Studies on experimental concussion: relation of physiologic effect to time duration of intracranial pressure increase at impact.</a> Neurology. 4, 674-681 (1954).</li><li>Hellmich, H. L., Capra, B., Eidson, K., Garcia, J., Kennedy, D., Uchida, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+neuronal+injury+after+traumatic+brain+injury.">Dose-dependent neuronal injury after traumatic brain injury.</a> Brain Research. 1044, 144-154 (2005).</li><li>Bramlett, H. M., Detrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+consequences+of+Traumatic+Brain+Injury:+Current+Status+of+Potential+Mechanisms+of+Injury+and+Neurological+Outcomes.">Long-Term consequences of Traumatic Brain Injury: Current Status of Potential Mechanisms of Injury and Neurological Outcomes.</a> J. Neurotrauma. 32, 1-15 (2015).</li><li>Pierce, J. E. S., Smith, D. H., Trojanowski, J. Q., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enduring+cognitive+neurobehavioral+and+histopathological+changes+persist+for+up+to+one+year+following+severe+experimental+brain+injury+in+rats.">Enduring cognitive neurobehavioral and histopathological changes persist for up to one year following severe experimental brain injury in rats.</a> Neuroscience. 87 (2), 359-369 (1998).</li><li>Smith, D. H., Chen, X. H., Pierce, J. E. S., Wolf, J. A., Trojanowski, J. Q., Graham, D. I., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+atrophy+and+neuron+death+for+one+year+following+brain+trauma+in+the+rat.">Progressive atrophy and neuron death for one year following brain trauma in the rat.</a> J Neurotrauma. 14 (10), 715-727 (1997).</li><li>Schallert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+tests+for+preclinical+intervention+assessment.">Behavioral tests for preclinical intervention assessment.</a> NeuroRx. 3 (4), 497-504 (2006).</li><li>Sell, S. L., Avila, M. A., Yu, G., Vergara, L., Prough, D. S., Grady, J. J., DeWitt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+resuscitation+improves+neuronal+and+behavioral+outcomes+after+traumatic+brain+injury+plus+hemorrhage.">Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage.</a> Anesthesiology. 108 (5), 873-881 (2008).</li><li>Hamm, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+assessment+of+outcome+following+traumatic+brain+injury+in+rats:+an+evaluation+of+selected+measures.">Neurobehavioral assessment of outcome following traumatic brain injury in rats: an evaluation of selected measures.</a> J Neurotrauma. 18 (11), 1207-1216 (2001).</li><li>Feeney, D. M., Gonzalez, A., Law, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine,+haloperidol,+and+experience+interact+to+affect+rate+of+recovery+after+motor+cortex+injury.">Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury.</a> Science. 217, 855-857 (1982).</li><li>Hamm, R. J., Temple, M. D., Pike, B. R., O'Dell, D. M., Buck, D. L., Lyeth, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+memory+deficits+following+traumatic+brain+injury+in+the+rat.">Working memory deficits following traumatic brain injury in the rat.</a> J Neurotrauma. 13, 317-323 (1996).</li><li>Morris, R. G., Hagan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allocentric+spatial+learning+by+hippocampectomised+rats:+A+further+test+of+the+"Spatial+Mapping"+and+"Working+Memory"+Theories+of+hippocampal+function.">Allocentric spatial learning by hippocampectomised rats: A further test of the "Spatial Mapping" and "Working Memory" Theories of hippocampal function.</a> The Quart J of Exp Psych. 38 (4), 365-395 (1986).</li><li>Dixon, C. E., Lyeth, B. G., Povlishock, J. T., Findling, R. L., Hamm, R. J., Marmarou, A., Young, H. F., Hayes, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluid-percussion+model+of+experimental+brain+injury+in+the+rat.">A fluid-percussion model of experimental brain injury in the rat.</a> J. Neurosurg. 67, 110-119 (1987).</li><li>DeWitt, D. S., Smith, T. G., Deyo, D. J., Miller, K. R., Uchida, T., Prough, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-arginine+and+superoxide+dismutase+prevent+or+reverse+cerebral+hypoperfusion+after+fluid-percussion+traumatic+brain+injury.">L-arginine and superoxide dismutase prevent or reverse cerebral hypoperfusion after fluid-percussion traumatic brain injury.</a> J. Neurotrauma. 14, 223-233 (1997).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A Language and Environment for Statistical Computing. , (2017).</li><li>Pohlert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Pairwise Multiple comparison of Mean Ranks Package (PMCMR) R package. , (2014).</li><li>Verma, P., Hellemans, K. G., Choi, F. Y., Yu, W., Weinberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+phase+and+sex+effects+on+depressive/anxiety-like+behaviors+and+HPA+axis+responses+to+acute+stress.">Circadian phase and sex effects on depressive/anxiety-like behaviors and HPA axis responses to acute stress.</a> Physiol Behav. 99, 276-285 (2010).</li><li>Schallert, T., Woodlee, M. T., Fleming, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Focal+Ischemic+Injury:+Behavior-Brain+Interactions+and+Issues+of+Animal+Handling+and+Housing.">Experimental Focal Ischemic Injury: Behavior-Brain Interactions and Issues of Animal Handling and Housing.</a> ILAR J. 44 (2), 130-143 (2003).</li><li>Ruis, J. F., Rietveld, W. J., Buys, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+parametric+photic+entrainment+of+circadian+rhythms+in+the+rat.">Properties of parametric photic entrainment of circadian rhythms in the rat.</a> Physiol Behav. 50, 1233-1239 (1991).</li><li>Ferguson, S. A., Maier, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+seasonal/circannual+effects+of+laboratory+rodent+behavior.">A review of seasonal/circannual effects of laboratory rodent behavior.</a> Physiol Behav. 119, 130-136 (2003).</li><li>Fujimoto, S. T., Longhi, L., Saatman, K. E., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+cognitive+function+evaluation+following+experimental+traumatic+brain+injury.">Motor and cognitive function evaluation following experimental traumatic brain injury.</a> Neurosci &amp; Biobehav Reviews. , (2004).</li><li>Gold, E. M., Su, D., Lopez-Velazquez, L., Haus, D. L., Perez, H., Lacuesta, G. A., Anderson, A. J., Cummings, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+long-term+deficits+in+rodent+models+of+traumatic+brain+injury.">Functional assessment of long-term deficits in rodent models of traumatic brain injury.</a> Regen. Med. 8 (4), 483-516 (2013).</li></ol>

The authors have nothing to disclose.

When conducting any type of behavioral testing, it is critical to be consistent. This detail includes many considerations that seem insignificant but have a major impact on the response of the animal. An important step that cannot be overlooked is acclimation of animals to their home-cage/housing situation prior to any experiment. This preparation reduces the effects of the animals&#39; physiological stress response, which can alter behavioral outcomes18. Similarly, it is absolutely essential that every effort is made to handle all animals in the same manner. This consistency includes, as previously mentioned, acclimation to housing and also acclimation to handling and transportation between rooms prior to training or testing. This concept cannot be overstated. Sloppy animal handling is disastrous to any behavioral testing19. Likewise, every effort needs to be made to test animals at the same time of day, whether during their dark or light cycle. For the tests discussed here, testing during light or dark stage is acceptable, as long as the tests are performed consistently. Testing done at different times during the circadian cycle has been shown to alter behavioral outcomes18,20. Additionally, the handler as well as the animal must be in a stress free, calm state in order to maximize the accuracy of results.
Particularly in the case of the Neuroscore, false positives and negatives are common. False positives typically occur when an animal is not fully habituated to handling and testing. The animal must be completely relaxed so the observed response is reflexive and not due to muscles tightening from reacting out of stress or fear. A tense handler can influence the results by transmitting stress to the animal. Therefore, holding the rat too tight or too loose can both be problematic. Additionally, if the handler is nervous, this can confound the reaction of the rats. There is also the risk that an inexperienced observer will misinterpret the rat&#39;s response. Good training and a lot of practice are essential to the success and consistency of the Neuroscore.
In general, the chief concern with these tests is the lack of a large difference, and sometimes no difference, between treatment groups. Since animals can react differently to different handlers, noises, times of day, and potentially, seasons21, every effort must be made to reduce all possible confounding factors.
The results of the Beam-Balance and Beam-Walk tasks shown here demonstrate that these tests are useful early after injury to detect deficits in vestibulomotor function. These deficits typically resolve over time1,14. In this model, by 6 months after injury, the injury-induced deficits have resolved. The results of the 6 month time point indicate that there are no differences between NAIVE, SHAM, or injured rats; however, all the rats have been relaxing in their home cages for 6 months, aging and gaining weight. Thus, by the time they are re-tested at 6 months post-surgery (or equivalent in the case of NA&#207;VE), they are essentially becoming old and fat, and therefore all the groups do not perform as well as they did compared to their baseline Day 0 results.
Another important consideration is that the behavior test used is the correct test. For example, the tests employed here are thought to represent the function of specific brain areas. One example is the vestibular system, which is important for balance. Brain areas involved in sensorimotor function, such as the cortex including sensorimotor cortex, the thalamus, corticospinal neurons, basal ganglia, nigro-striatum, to name a few, are all involved in vestibulomotor coordination. Thus, deficits in the Beam-Balance or Beam-Walk indicate potential deficits in these areas. Furthermore, the hippocampus and prefrontal cortex are involved in the learning and memory functions tested by the working memory water maze. Even when the correct test is chosen, the limitations of the tests that are employed must be kept in mind. For example, none of the tests presented here are sensitive to deficits in mood, such as depression, anxiety, or social interactions such as aggression, decision making, or impulsivity. To reiterate, it is imperative to choose the appropriate test for the behavior and brain area to be evaluated.
Interpretation and analysis of behavioral data must be approached with caution. It is highly recommended to include power analyses of each type of test separately, because, using a behavioral outcome as a measure of a neural deficit, is by its nature, a crude measure of a subtle effect. Furthermore, different tests require different types of statistical analyses. For example, the Neuroscore and Beam-Balance tests described are dependent on the interpretation of a trained observer to score the behavior using an ordinal scale. These types of data are not continuous and not normally distributed, so nonparametric statistics should be used, such as the Kruskal-Wallis test, as demonstrated in Sections 6.1 and 6.2. Alternatively, the Beam-Walk and working memory water maze tests produce data that are continuous and normally distributed, so parametric statistics can be used, such as one-way ANOVA or repeated-measures two-way ANOVA, as demonstrated in Sections 6.3 and 6.4.
The behavioral tasks presented here have stood the test of time and give reproducible results, particularly when paired with the FPI model in rats, although many other methods of behavioral testing for brain injury do exist. The neuroscore is a short assessment performed with a minimum of equipment. Other tests of reflexes and strength are available and could be incorporated into a neurological assessment, such as the lateral pulsion task, the akinesia test, the inclined plane test, and grip strength (see Fujimoto et al.22 and Gold et al.23). The Beam-Balance and Beam-Walk tasks described are measures of vestibulomotor deficits after injury. Vestibulomotor coordination can be considered one measure of gross locomotor behavior, while other measures of gross locomotor deficits include the Rotarod, the rotating pole, and open field activity. The ability to swim, measured as swim speed during the water maze, is also an indication of gross motor coordination22,23. The working memory water maze task completes this set of tests by detecting both reference memory deficits (indicated by Trial 1) and working memory deficits (indicated by Trial 2 or the difference between Trial 1 and Trial 2). Other measures of cognitive function include the eight arm radial maze, the Barnes maze, the novel object recognition test, and different variations of the water maze. These variations include the original Morris water maze and the Lashley III maze (again see Fujimoto et al.22 and Gold et al.23). This battery of tests has proven to be useful early after injury and, in varying degrees, out to 12 months after injury1.
Additionally, the tasks demonstrated here can be used with different strains, sex, and ages of rats; however, accommodations may need to be made for differing sizes and in cases of greater frailty. For example, older, heavier rats need a wider beam for the Beam-Balance task and aged, frail rats, may need shorter duration of swim times in the water maze. Thus, there is room for flexibility in these tests and potential for development of new tests to accommodate different situations and hypotheses.

The methods presented here are designed to detect functional deficits in specific brain areas induced by an experimental model of TBI in the rat. Four different behavior tests will be described. First, the short neurological assessment, referred to as the neuroscore, can be performed without requiring any specialized equipment but does require practice; this test detects deficits in reflexes. Second, the Beam-Balance test detects deficits in the ability to balance. This task requires the handler to score the rat based on an ordinal scale and requires some training of the handler. The Beam-Balance test requires a narrow beam and is sensitive to deficits in the vestibular system. The third test assesses vestibulomotor coordination. This test is known as the Beam-Walk task, and although some pre-training of the rat is required, this procedure is more objective than the previous two as the latency to traverse the beam is an objective measure not dependent on subjective scoring. This difference is because the time to traverse a narrow beam to reach a safe box is measured. The Beam-Walk test requires a longer beam than the Beam-Balance as well as an escape box. This test measures deficits in both motor coordination and balance and thus is sensitive to damage to the cerebellum and motor related brain areas. The working memory version of the Morris water maze (MWM-WM) primarily tests hippocampal function and integration with the prefrontal cortex or executive function. The version of the Morris water maze shown can also be used to detect deficits in reference memory1.
These methods were chosen based on their well-established track record in the literature. Each one has been effective in many hands from different laboratories with multiple strains of rats over numerous years of research. However, in the past, post-injury measures up to two weeks after injury were considered &#34;chronic&#34; time points. Thus, to establish behavioral techniques for the study of chronic effects of TBI in rodents, these well-known methods needed to be evaluated for sensitivity to detect TBI induced deficits at longer time points after injury. While there are now several rodent models of TBI, the FPI model is one of the most widely used, and is applied in this study. This model was first published in the 1950&#39;s2, and since then, more than 1,000 papers have employed FPI in rats3. The neuropathology of this type of injury has been well-described by us4 and others5,6,7. Briefly, neuronal injury in the hippocampus has been shown to be dose-dependent using Fluoro-Jade staining at short times after injury, i.e., 24-48 h; while gross atrophy and cavitation including thinning of the internal capsule and cortex has been reported at one year after injury6,7.
The most meaningful representation of brain function is assessed by using behavioral outcome measures after an experimental brain injury. However, the vast majority of FPI experiments that use behavioral outcomes make measures relatively early, typically from 1 to 14 days after injury. Using the methods demonstrated here, some behavioral deficits can be detected out to 12 months after injury1.Neurological function, gross vestibulomotor function, and fine motor coordination were assessed on post-injury days (PIDs) 1-3 and at 3, 6, and 12 months after surgery, using a short neurological assessment (Neuroscore; modified from Schallert8), the Beam-Balance task, and the Beam-Walk task9,10,11. Reference and working memory were assessed using a working memory version of the Morris water maze1,12,13.

<table><tbody><tr><td>Sprague-Dawley rats</td><td>Charles Rivers Laboratories&lt;br/&gt; 251 Ballardvale St&lt;br/&gt; Wilmington, MA 01887-1096&lt;br/&gt; Phone: 800-522-7287</td><td>CD-IGS rats, strain code 001</td><td>male, albino, 300-350g at arrival</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Beam-Balance&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beam</td><td>home built</td><td></td><td>wood, 25&quot; l x 1&quot; h x 3/4&quot; w sealed with polyurethane varnish</td></tr><tr><td>C-clamp</td><td>Home Depot</td><td>1422-C</td><td>2 1/2&quot;</td></tr><tr><td>barrier</td><td>Home Depot</td><td></td><td>styrofoam, 18&quot; x 17 1/2&quot;</td></tr><tr><td>table (for both BB &amp;amp; BW)</td><td>generic office supply</td><td></td><td>37&quot; h x 30&quot; w x 60&quot; l</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Beam-Walk&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beam</td><td>home built</td><td></td><td>wood 38-1/2&quot; l x 1-3/4&quot; h x 1&quot; w sealed with polyurethane varnish (~ 37&quot; off floor)</td></tr><tr><td>escape box</td><td>home built</td><td></td><td>woodpainted black 12 1/2 &quot; l x 9&quot; h x 7-1/4&quot; w</td></tr><tr><td>nails (pegs)</td><td></td><td></td><td>2&quot;</td></tr><tr><td>hinges</td><td></td><td></td><td></td></tr><tr><td>clamps</td><td></td><td></td><td></td></tr><tr><td>white noise machine</td><td>San Diego Instruments&lt;br/&gt; 9155 Brown Deer Rd, Suite 8 San Diego, CA 92121&lt;br/&gt; Phone: (858)530-2600</td><td></td><td>http://www.sandiegoinstruments.com/libraries/misc/datasheets/whitenoise.pdf</td></tr><tr><td>light</td><td>Home Depot</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Morris Water Maze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>fiberglass pool</td><td>manufacturer unknown</td><td></td><td></td></tr><tr><td>(similar to one made by SDI)</td><td>San Diego Instruments</td><td>7000-0723</td><td>72&quot; diameter x 30&quot; deep (~ 500 gal)</td></tr><tr><td>plexiglass platform</td><td>hand-made by Maggie Parsley</td><td></td><td>10 cm diameter, 26&quot; tall with silicone applied to the surface of the platform to provide a gripping surface</td></tr><tr><td>(similar to one made by SDI)</td><td>SDI</td><td>7500-0272</td><td></td></tr><tr><td>plexiglass animal boxes w/ lids</td><td>UTMB Machine Shop</td><td></td><td>2 boxes, 10&quot; w x 16&quot; L x 9&quot; h</td></tr><tr><td>spot lights/ heat lamps</td><td>Home Depot</td><td></td><td>3 around pool, 2 over boxes to dry animals</td></tr><tr><td>AnyMaze</td><td>San Diego Instruments&lt;br/&gt; 9155 Brown Deer Rd, Suite 8 San Diego, CA 92121&lt;br/&gt; Phone: (858)530-2600</td><td>/9001</td><td>http://www.sandiegoinstruments.com/any-maze-video-tracking/</td></tr></tbody></table>

detecting behavioral deficits in rats after traumatic brain injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

Watch this Scientific Journal Video about Detecting Behavioral Deficits in Rats After Traumatic Brain Injury at JoVE.com

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

detecting-behavioral-deficits-in-rats-after-traumatic-brain-injury

Universidad San Sebastian

Research

JoVE Journal

Behavior

17.8K Views.  University of Texas Medical Branch. The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

Video: Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Neuroscience

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

Medicine

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social hierarchies that are often based on dominant-submissive relationships. The dominant-submissive relationship is a normal behavioral pattern among the individuals of a species. Traumatic brain injury is a frequent cause of social interaction impairment and the reorganization of dominant-submissive relationships in animal pairs. This protocol describes submissive behavior in adult male Sprague-Dawley rats after the induction of traumatic brain injury using a fluid-percussion model compared to naive rats through a series of dominant-submissive tests performed between 29 days and 33 days after induction. The dominant-submissive behavior test shows how brain injury can induce submissive behavior in animals competing for food. After traumatic brain injury, the rodents were more submissive, as indicated by them spending less time at the feeder and being less likely to arrive first at the trough compared to the control animals. According to this protocol, submissive behavior develops after traumatic brain injury in adult male rats.

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social ...

Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Bioengineering

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...