Animal Spinal Cord Injury Study Research Articles

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury

BackgroundSpinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI.MethodsA contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro.ResultsStepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation.ConclusionsOur findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.Graphical

Glutathione effect on functional and histological recovery after spinal cord injury in rats

ObjectiveThe aim of this study was to evaluate the GSH effect on functional and histological recovery after experimental spinal cord injury in rats. MethodsForty Wistar rats were subjected to spinal cord injury through the Multicenter Animal Spinal Cord Injury Study (MASCIS) Impactor system. The rats were sorted and divided into four groups, as follows: Group 1 ‒ Laminectomy and spinal cord injury; Group 2 ‒ Laminectomy, spinal cord injury and Saline Solution (SS) 0.9%; Group 3 ‒ Laminectomy, spinal cord injury, and GSH; and Group 4 ‒ lLaminectomy without spinal cord injury. GSH and SS were administered intraperitoneally. Groups 1 and 4 received no intervention. ResultsThe rats were evaluated for locomotor function recovery at seven different times by the Basso, Beattie, and Bresnahan (BBB) scale on days 2, 7, 14, 21, 28, 35, and 42 after the spinal cord injury. On day 42, the rats were sacrificed to analyze the histological findings of the injured spinal cord. In the group submitted to GSH, our experimental study revealed better functional scores on the BBB scale, horizontal ladder scale, and cranial and caudal axon count. The differences found were statistically significant in BBB scores and axonal count analysis. ConclusionThis study demonstrated that using glutathione in experimental spinal trauma can lead to better functional recovery and improved axonal regeneration rate in Wistar rats submitted to experimental spinal cord injury.

Beneficial Effects of Ethanolic Extract of the Medicinal Herb Mucuna Pruriens Against Oxidative Stress and Inflammation Might be Limited in Contusive Spinal Cord Injury

Experimental studies found extracts of Mucuna pruriens (L.) DC, a plant used in the traditional medical systems to possess strong anti-inflammatory and anti-oxidant properties among a wide range of other beneficial effects. Hypothesizing the possibility for a multifaceted beneficial effect, the therapeutic potential of the ethanolic extract of Mucuna pruriens (MP) in treating spinal cord injury (SCI) was tested using the highly standardized Multicenter Animal Spinal Cord Injury Study (MASCIS) animal model of contusive SCI. Male Wister rats with SCI were treated with the ethanolic extract of MP at a dose of 200 mg/kg/day for 10 weeks. The outcome was assessed through molecular, biochemical, histological, and behavioral scoring parameters. Results indicated anti-apoptotic, anti-necrotic, anti-inflammatory, and neuroprotective effects of MP. Nevertheless, many of these beneficial effects were not statistically significant and there was no functional improvement due to MP treatment. MP at comparable doses was reported to be significantly effective in mitigating oxidative stress and/or inflammation under various other pathological conditions. Lack of significance in the present study could be due to the presence of blood-spinal cord-barrier; that might have prevented the components of MP from reaching spinal cord tissue in adequate quantities. Additionally, heterogeneity in the function of the cells typical to the CNS environment could be the reason for the failure of recovery. To conclude, the limitations posed by the structural and functional distinctions in the spinal cord environment in comparison to other non-CNS tissue environments deserve attention while adjudicating the efficacies of herbal remedies for SCI.

Excavating FAIR Data: the Case of the Multicenter Animal Spinal Cord Injury Study (MASCIS), Blood Pressure, and Neuro-Recovery

Meta-analyses suggest that the published literature represents only a small minority of the total data collected in biomedical research, with most becoming ‘dark data’ unreported in the literature. Dark data is due to publication bias toward novel results that confirm investigator hypotheses and omission of data that do not. Publication bias contributes to scientific irreproducibility and failures in bench-to-bedside translation. Sharing dark data by making it Findable, Accessible, Interoperable, and Reusable (FAIR) may reduce the burden of irreproducible science by increasing transparency and support data-driven discoveries beyond the lifecycle of the original study. We illustrate feasibility of dark data sharing by recovering original raw data from the Multicenter Animal Spinal Cord Injury Study (MASCIS), an NIH-funded multi-site preclinical drug trial conducted in the 1990s that tested efficacy of several therapies after a spinal cord injury (SCI). The original drug treatments did not produce clear positive results and MASCIS data were stored in boxes for more than two decades. The goal of the present study was to independently confirm published machine learning findings that perioperative blood pressure is a major predictor of SCI neuromotor outcome (Nielson et al., 2015). We recovered, digitized, and curated the data from 1125 rats from MASCIS. Analyses indicated that high perioperative blood pressure at the time of SCI is associated with poorer health and worse neuromotor outcomes in more severe SCI, whereas low perioperative blood pressure is associated with poorer health and worse neuromotor outcome in moderate SCI. These findings confirm and expand prior results that a narrow window of blood-pressure control optimizes outcome, and demonstrate the value of recovering dark data for assessing reproducibility of findings with implications for precision therapeutic approaches.

Does timing of transplantation of neural stem cells following spinal cord injury affect outcomes in an animal model?

We previously reported that functional recovery of rats with spinal cord contusions can occur after acute transplantation of neural stem cells distal to the site of injury. To investigate the effects of timing of administration of human neural stem cell (hNSC) distal to the site of spinal cord injury on functional outcomes in an animal model. Thirty-six adult female Long-Evans hooded rats were randomized into three experimental and three control groups with six animals in each group. The T10 level was exposed via posterior laminectomy, and a moderate spinal cord contusion was induced by the Multicenter Animal Spinal Cord Injury Study Impactor (MASCIS, W.M. Keck Center for Collaborative Neuroscience, Piscataway, NJ, USA). The animals received either an intrathecal injection of hNSCs or control media through a separate distal laminotomy immediately, one week or four weeks after the induced spinal cord injury. Observers were blinded to the interventions. Functional assessment was measured immediately after injury and weekly using the Basso, Beattie, Bresnahan (BBB) locomotor rating score. A statistically significant functional improvement was seen in all three time groups when compared to their controls (acute, mean 9.2 vs. 4.5, P=0.016; subacute, mean 11.1 vs. 6.8, P=0.042; chronic, mean 11.3 vs. 5.8, P=0.035). Although there was no significant difference in the final BBB scores comparing the groups that received hNSCs, the group which achieved the greatest improvement from the time of cell injection was the subacute group (+10.3) and was significantly greater than the chronic group (+5.1, P=0.02). The distal intrathecal transplantation of hNSCs into the contused spinal cord of a rat led to significant functional recovery of the spinal cord when injected in the acute, subacute and chronic phases of spinal cord injury (SCI), although the greatest gains appeared to be in the subacute timing group.

Systemic Administration of Connexin43 Mimetic Peptide Improves Functional Recovery after Traumatic Spinal Cord Injury in Adult Rats

Blocking of Connexin43 hemichannels, the main gap junction protein located on astrocytes in the central nervous system, has been shown to reduce neural injury in a number of models. We demonstrated previously that local administration of a Connexin43 mimetic peptide, Peptide5, reduces secondary tissue damage after spinal cord injury (SCI). Here, we investigated whether acute systemic delivery of Peptide5 is also protective in a model of SCI. Rats were subjected to a mild spinal cord contusion using the Multicentre Animal Spinal Cord Injury Study impactor and were injected intraperitoneally with Peptide5 or a scrambled peptide immediately and at 2 h and 4 h post-injury. Rats were tested for locomotor recovery and pain hypersensitivity and euthanized at 8 h, 24 h, two weeks, or six weeks post-injury. Compared with control rats, Peptide5 treated rats showed significant improvement in hindlimb locomotor function between three and six weeks post-injury and reductions in at-level mechanical allodynia at weeks one and six post-injury. Immunohistochemistry showed that Peptide5 treatment led to a reduction in total Connexin43 and increased phosphorylated Connexin43 at 8 h compared with scrambled peptide. At two and six weeks, lesion size, the astrocytic and the activated macrophage, and/or microglial response were all decreased in the Peptide5 animals. In addition, neuronal cell numbers were higher in the Peptide5 animals compared with the scrambled peptide treated rats at two and six weeks. These results show for the first time that systemic administration of Peptide5 to block the pathological opening of Connexin43 hemichannels is a feasible treatment strategy in this setting, ameliorating the secondary SCI.

Local versus distal transplantation of human neural stem cells following chronic spinal cord injury

Background ContextPrevious studies have demonstrated functional recovery of rats with spinal cord contusions after transplantation of neural stem cells adjacent to the site of acute injury. PurposeThe purpose of the study was to determine if the local or distal injection of neural stem cells can cause functional difference in recovery after chronic spinal cord injury. Study Design/SettingTwenty-four adult female Long-Evans hooded rats were randomized into four groups, with six animals in each group: two experimental and two control groups. Functional assessment was measured after injury and then weekly for 6 weeks using the Basso, Beattie, and Bresnahan locomotor rating score. Data were analyzed using two-sample t test and linear mixed-effects model analysis. MethodsPosterior exposure and laminectomy at the T10 level was used. Moderate spinal cord contusion was induced by the Multicenter Animal Spinal Cord Injury Study Impactor with 10-g weight dropped from a height of 25 mm. Experimental subjects received either a subdural injection of human neural stem cells (hNSCs) locally at the injury site or intrathecal injection of hNSCs through a separate distal laminotomy 4 weeks after injury. Controls received control media injection either locally or distally. ResultsA statistically significant functional improvement in subjects that received hNSCs injected distally to the site of injury was observed when compared with the control (p=.042). The difference between subjects that received hNSCs locally and the control did not reach statistical significance (p=.085). ConclusionsThe transplantation of hNSCs into the contused spinal cord of a rat led to significant functional recovery of the spinal cord when injected distally but not locally to the site of chronic spinal cord injury.

Contusive spinal cord injury up regulates mu-opioid receptor (mor) gene expression in the brain and down regulates its expression in the spinal cord: possible implications in spinal cord injury research

Traumatic spinal cord injury (SCI) is one of the dreaded neurological conditions and finding a cure for it has been a hot area of research. Naloxone – a mu-opiate receptor (mor) antagonist was considered for SCI treatment based on its positive effects under shock conditions. In contrary to animal studies based reports about the potential benefits of naloxone in treating SCI, a large scale clinical trial [National Acute Spinal Cord Injury Study II (NASCIS II)] conducted in USA failed to witness any effectiveness. The inconsistency noticed was intriguing. Therefore, the objective of the present study was to re-examine the role of naloxone in treating SCI using a highly standardised Multicenter Animal Spinal Cord Injury Study (MASCIS) animal model of contusive SCI. Results indicated that naloxone produced negligible and insignificant neuroprotection. In an attempt to understand the cause for the failure, it was found that mu-opioid receptor (mor) gene expression was upregulated in the brain but was down regulated in the spinal cord after contusive SCI. Given that the beneficial effects of naloxone are through its action on the mor, the results indicate that unlike the brain, spinal cord might not be bracing to utilise the opiate system in the repair process. This could possibly explain the failure of naloxone treatment in NASCIS II. To conclude, opiate antagonists like naloxone may be neuroprotective for treating traumatic brain injuries, but not for traumatic/contusive spinal cord injuries.

An experimental spinal cord injury rat model using customized impact device: A cost-effective approach

Till date, NYU MASCIS (New York University, Multicenter Animal Spinal Cord Injury Study) impactor and Ohio State University electromagnetic spinal cord injury device impactor were under use for simulating an experimental spinal cord injury in rodents; functional recovery being assessed through Basso, Beattie and Bresnahan (BBB) scoring method which is an open field behavior based scoring system. Although, the cited impactors are state-of-art devices, affordability to scientists in developing and under developed countries is questionable. Since the acquisition of these impact devices are expensive, we designed a customized impact device based on the requirement, satisfying all the parameters to withstand a standard animal model for contusion type of spinal cord injury at the thoracic level without compromising the lesion reproducibility. Here, a spinal cord contusion is created using a blunt-force impactor in male Wistar rats. Our method gave consistent lesion effects as evaluated by behavior scoring methods. All the animals showed equal degree of performance in tests like narrow beam, inclined plane and horizontal ladder and in BBB scores (open field locomotor test). The aim of presenting our experience is to reinstate the fact that lack of affordability to get sophisticated instrumentation need not be a hurdle in the pursuit of science.

Functional assessment of the acute local and distal transplantation of human neural stem cells after spinal cord injury

Spinal cord injury can lead to severe functional impairments secondary to axonal damage, neuronal loss, and demyelination. The injured spinal cord has limited regrowth of damaged axons. Treatment remains controversial, given inconsistent functional improvement. Previous studies demonstrated functional recovery of rats with spinal cord contusion after transplantation of rat fetal neural stem cells. We hypothesized that acute transplantation of human fetal neural stem cells (hNSCs) both locally at the injury site as well as distally via intrathecal injection would lead to improved functional recovery compared with controls. Twenty-four adult female Long-Evans hooded rats were randomized into four groups with six animals in each group: two experimental and two control. Functional assessment was measured after injury and then weekly for 6 weeks using the Basso, Beattie, and Bresnahan Locomotor Rating Score. Data were analyzed using two-sample t test and linear mixed-effects model analysis. Posterior exposure and laminectomy at T10 level was used. Moderate spinal cord contusion was induced by the Multicenter Animal Spinal Cord Injury Study Impactor with 10-g weight dropped from a height of 25 mm. Experimental subjects received either a subdural injection of hNSCs locally at the injury site or intrathecal injection of hNSCs through a separate distal laminotomy. Controls received control media injection either locally or distally. Statistically significant functional improvement was observed in local or distal hNSCs subjects versus controls (p=.034 and 0.016, respectively). No significant difference was seen between local or distal hNSC subjects (p=.66). Acute local and distal transplantation of hNSCs into the contused spinal cord led to significant functional recovery in the rat model. No statistical difference was found between the two techniques.

Modelo experimental de lesão medular e protocolo de avaliação motora em ratos wistar

OBJETIVOS: Determinar modelo de contusão medular padronizado e método de avaliação motora em ratos com quadro de paraplegia. MÉTODOS: Utilizados 20 ratos Wistar divididos em quatro grupos, diferenciado o nível de gravidade da lesão raquimedular; realizadas lesões intermediárias padronizadas, através do sistema "MASCIS IMPACTOR" (Multicenter Animal Spinal Cord Injury Study): grupo 1 - 12,5 mm (lesão leve); grupo 2 - 25 mm (lesão moderada); grupo 3 - 50 mm (lesão grave); grupo 4 - animais que não sofreram lesão (grupo controle). Após 48 horas foi avaliada função motora, segundo escala proposta por Basso, Beattie e Bresnahan. RESULTADOS: Utilizando o modelo, notamos que as contusões leves (12,5mm de altura) foram efetivas, e os animais apresentaram infecção urinária na fase aguda uma semana após a contusão. As contusões intermediárias (25 mm de altura) foram efetivas, os animais apresentaram infecção urinária até duas semanas após a contusão. As contusões graves (50mm de altura) foram efetivas, os animais apresentaram infecção urinária durante três a quatro semanas e autofagia. CONCLUSÃO: O modelo de lesão medular utilizando o sistema MASCIS IMPACTOR e a avaliação funcional proposta por Basso, Beattie e Bresnahan é reprodutível, podendo ser utilizada, propiciando troca de informações entre os diferentes pesquisadores.

Cannabinoid subtype-2 receptors modulate the antihyperalgesic effect of WIN 55,212-2 in rats with neuropathic spinal cord injury pain

Background context There is increasing evidence for a role of the cannabinoid (CB) system in the development of neuropathic pain (NP) after spinal cord injury (SCI). The nonspecific CB 1 and CB 2 receptor agonists, WIN 55, 212-2 (WIN), have previously been shown to alleviate both mechanical and thermal hyperalgesia (TH) after peripheral nerve injury. Purpose The present study was designed to identify the CB receptors involved in the antihyperalgesic effect of WIN by using selective antagonists for CB 1 and CB 2 receptors. Study design This is an in vivo and behavioral study using a moderate T9 contusion SCI. After injury, TH of the hind paws was measured on postinjury days 21 through 42. Methods Sprague-Dawley rats underwent a contusion SCI using the Multicenter Animal Spinal Cord Injury Study (MASCIS) weight-drop impactor, which induced a moderate T9 SCI. Only animals showing consistent plantar stepping and consistent forelimb and hind limb coordination (Basso, Beattie, and Bresnahan score=15) were tested for TH. Animals exhibiting decreased withdrawal latency time, indicating TH, on or before Day 42, were selected for pharmacological intervention. Animals not exhibiting TH did not receive pharmacological intervention and were sacrificed. Rats underwent hind paw testing before any drug administration (after injury), 45 minutes after selective CB antagonist (AM 251 or AM 630) administration (postantagonist) and again 45 minutes after WIN administration (post-WIN). There were a total of seven treatment groups: saline vehicle control; Dimethyl sulfoxide (DMSO) vehicle control; low-dose WIN (0.2 mg/kg); and high-dose WIN (2.0 mg/kg); AM 251 (3 mg/kg) and AM 630 (1 mg/kg) were given subcutaneously in a total volume of 0.5 mL. Followed by intraperitoneal injection of WIN after each antagonist, sham-operated rats repeated pharmacological intervention used with treatment Groups 5 and 6. Results Thermal hyperalgesia was significantly ameliorated in a dose-dependent manner with systemically administered WIN. Cannabinoid receptor Type 1 antagonist AM 251 pretreatment did not affect the antihyperalgesic effect of WIN. By contrast, pretreatment with the CB 2 receptor antagonist AM 630 significantly attenuated the effect of WIN. Conclusion Taken together, these results suggest a role of the CB 2 receptor in modulating SCI-induced TH. Selective activation of the CB 2 receptor could potentially lead to analgesic effects on NP while avoiding psychotropic side effects in patients with SCI.

Characterization of Graded Multicenter Animal Spinal Cord Injury Study Contusion Spinal Cord Injury Using Somatosensory-Evoked Potentials

Electrophysiological analysis using somatosensory-evoked potentials (SEPs) and behavioral assessment using Basso, Beattie, Bresnahan (BBB) scale were compared over time for graded Multicenter Animal Spinal Cord Injury Study (MASCIS) contusion spinal cord injury (SCI). To study the SEP responses across different contusion injury severities and to compare them with BBB scores. For any SCI therapy evaluation, it is important to accurately and objectively standardize the injury model. The graded MASCIS contusion injuries on dorsal spine have been standardized using BBB, which is subjective and prone to human errors. Furthermore, dorsal pathway disruption does not always produce locomotor deficits. SEP monitoring provides an advantage of providing a reliable and objective assessment of the functional integrity of dorsal sensory pathways. Four groups of Fischer rats received contusion at T8 using New York University (NYU)-MASCIS impactor from impact heights of 6.25 mm (mild), 12.5 mm (moderate), 25 mm (severe), or 50 mm (very severe). The control group underwent laminectomy only. SEP and BBB recordings were performed once before injury, and then weekly for up to 7 weeks. Graded levels of injury produced concomitant attenuations in hindlimb SEP amplitudes. Following injury, 25 and 50 mm groups together differed significantly from 12.5 and 6.25 mm groups (P < 0.01). From week 5, differences between 12.5 and 6.25 mm groups also became apparent (P < 0.01), which showed significant electrophysiological improvement. However, no significant differences were observed between 25 and 50 mm groups, which showed negligible electrophysiological recovery. Although comparable differences between different groups were also detected by BBB after injury (P < 0.001), BBB was less sensitive in detecting any improvement in 6.25 and 12.5 mm groups. SEP amplitudes and BBB scores decrease corresponding to increase in injury severity, however, these show different temporal patterns of recovery. These results demonstrate the utility of SEPs in conjunction with BBB, to monitor therapeutic interventions in SCI research.

Padronização da lesão de medula espinal em ratos Wistar

OBJETIVO: Padronizar um modelo experimental de lesão de medula espinal em ratos Wistar, utilizaram-se um equipamento computadorizado para impacto por queda de peso e os parâmetros determinados pelo Multicenter Animal Spinal Cord Injury Study - MASCIS. MÉTODOS: Avaliaram-se 30 ratos, com idade variando entre 20 e 25 semanas de vida. O peso variou de 200 a 300 g, para as fêmeas, e de 232 a 430 g para os machos. Realizaram-se impactos com pesos de 10 g de 12,5; 25 e 50 mm de altura, controlando-se a velocidade de impacto e o coeficiente de compressão. O impacto ocorreu sobre a superfície da medula espinal na altura da décima vértebra torácica, após laminectomia. Monitoraram-se os sinais vitais e realizaram-se gasometrias previamente e posteriormente à lesão da medula. O volume de lesão foi avaliado pela análise quantitativa dos íons de sódio e potássio. RESULTADOS: Verificaram-se correlações estatisticamente significantes entre o volume de lesão e os parâmetros mecânicos. O volume de lesão provocado por queda de 50 mm de altura foi superior aos de 12,5 e 25 mm, que não diferiram entre si. CONCLUSÃO: O modelo demonstrou-se eficaz e capaz de gerar lesões medulares padronizadas em ratos Wistar.

Endogenous repair after spinal cord contusion injuries in the rat.

Contusion injuries of the rat thoracic spinal cord were made using a standardized device developed for the Multicenter Animal Spinal Cord Injury Study (MASCIS). Lesions of different severity were studied for signs of endogenous repair at times up to 6 weeks following injury. Contusion injuries produced a typical picture of secondary damage resulting in the destruction of the cord center and the chronic sparing of a peripheral rim of fibers which varied in amount depending upon the injury magnitude. It was noted that the cavities often developed a dense cellular matrix that became partially filled with nerve fibers and associated Schwann cells. The amount of fiber and Schwann cell ingrowth was inversely related to the severity of injury and amount of peripheral fiber sparing. The source of the ingrowing fibers was not determined, but many of them clearly originated in the dorsal roots. In addition to signs of regeneration, we noted evidence for the proliferation of cells located in the ependymal zone surrounding the central canal at early times following contusion injuries. These cells may contribute to the development of cellular trabeculae that provide a scaffolding within the lesion cavity that provides the substrates for cellular infiltration and regeneration of axons. Together, these observations suggest that the endogenous reparative response to spinal contusion injury is substantial. Understanding the regulation and restrictions on the repair processes might lead to better ways in which to encourage spontaneous recovery after CNS injury.

MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study.

The Multicenter Animal Spinal Cord Injury Study (MASCIS) adopted a modified 21-point open field locomotor scale developed by Basso, Beattie, and Bresnahan (BBB) at Ohio State University (OSU) to measure motor recovery in spinal-injured rats. BBB scores categorize combinations of rat hindlimb movements, trunk position and stability, stepping, coordination, paw placement, toe clearance, and tail position, representing sequential recovery stages that rats attain after spinal cord injury. A total of 22 observers from 8 participating centers assessed 18 hindlimbs of 9 rats at 2-6 weeks after graded spinal cord injury. The observers were segregated into 10 teams. The teams were grouped into 3 cohorts (A, B, and C), consisting of one experienced team from OSU and two non-OSU teams. The cohorts evaluated the rats in three concurrent and sequential sessions. After viewing a rat for 4 min, individual observers first assigned scores without discussion. Members of each team then discussed and assigned a team score. Experience (OSU vs. non-OSU) and teamwork (individual vs. team) had no significant effect on mean scores although the mean scores of one cohort differed significantly from the others (p = 0.0002, ANOVA). However, experience and teamwork significantly influenced reliability of scoring. OSU team scores had a mean standard deviation or discordance of 0.59 points, significantly less than 1.31 points for non-OSU team scores (p = 0.003, ANOVA) and 1.30 points for non-OSU individual scores (p = 0.001, ANOVA). Discordances were greater at the upper and lower ends of the scale, exceeding 2.0 in the lower (< 5) and upper (> 15) ends of the scale but were < 1.0 for scores between 4 and 16. Comparisons of non-OSU and OSU team scores indicated a high reliability coefficient of 0.892 and a correlation index (r2) of 0.894. These results indicate that inexperienced observers can learn quickly to assign consistent BBB scores that approach those given by experienced teams, that the scores are most consistent between 4 and 16, and that experience improves consistency of team scores.