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J Neurosurg: Spine / Volume 17 / September 2012

J Neurosurg Spine (Suppl) 17:157–229, 2012

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DESPITE advances in the medical, surgical, and re-habilitation management of human SCI, there isno widely accepted treatment that attenuates the

complex biological processes that constitute the second-ary injury. One major category of treatment is neuropro-tection by pharmacotherapy offered in the acute and sub-

acute phases of injury and designed to protect spinal cordtissue from the severely damaging pathophysiologicalevents that occur in the CNS after physical trauma. Therehave been remarkable advances in our understanding ofthe secondary injury events after CNS trauma in both thebrain and spinal cord, and there are more than 25 second-ary injury processes that have been identied, offeringmultiple potential therapeutic opportunities to counteractthem.6,40,58,73 In the past 30 years a huge effort has beenexpended by clinicians, basic scientists, and industry todiscover effective neuroprotective agents for SCI, whichthus far have largely failed to improve recovery. There

Translational potential of preclinical trials of neuroprotectionthrough pharmacotherapy for spinal cord injury

CHARLES H. TATOR, M.D., PH.D., 1 ROBIN HASHIMOTO, PH.D., 2 ANNIE RAICH, M.P.H., 2 DANIEL NORVELL, PH.D., 2 MICHAEL G. FEHLINGS, M.D., PH.D., 1 JAMES S. HARROP, M.D., 3 JAMES GUEST, M.D., PH.D., 4 BIZHAN AARABI, M.D., F.R.C.S.C., 5 AND ROBERT G. GROSSMAN, M.D.6

1 Division of Neurosurgery and Spinal Program, Toronto Western Hospital and University of Toronto, Ontario,

Canada; 2Spectrum Research, Inc., Tacoma, Washington; 3 Department of Neurological Surgery, Thomas

Jefferson University, Philadelphia, Pennsylvania; 4 Department of Neurological Surgery and the Miami

Project to Cure Paralysis, Miller School of Medicine, University of Miami, Florida; 5 Department of

Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland; and 6 Department of

Neurosurgery, The Methodist Hospital, Houston, Texas

There is a need to enhance the pipeline of discovery and evaluation of neuroprotective pharmacological agentsfor patients with spinal cord injury (SCI). Although much effort and money has been expended on discovering effec-tive agents for acute and subacute SCI, no agents that produce major benet have been proven to date. The decien-cies of all aspects of the pipeline, including the basic science input and the clinical testing output, require examinationto determine remedial strategies. Where has the neuroprotective/pharmacotherapy preclinical process failed and whatneeds to be done to achieve success? These are the questions raised in the present review, which has 2 objectives:1) identication of articles that address issues related to the translational readiness of preclinical SCI pharmacologi-cal therapies; and 2) examination of the preclinical studies of 5 selected agents evaluated in animal models of SCI(including blunt force trauma, penetrating trauma, or ischemia). The 5 agents were riluzole, glyburide, magnesiumsulfate, nimodipine, and minocycline, and these were selected because of their promise of translational readiness asdetermined by the North American Clinical Trials Network Consortium.

The authors found that there are major deciencies in the effort that has been extended to coordinate and conductpreclinical neuroprotection/pharmacotherapy trials in the SCI eld. Apart from a few notable exceptions such as the

NIH effort to replicate promising strategies, this eld has been poorly coordinated. Only a small number of articleshave even attempted an overall evaluation of the neuroprotective/pharmacotherapy agents used in preclinical SCItrials. There is no consensus about how to select the agents for translation to humans on the basis of their preclinicalperformance and according to agreed-upon preclinical performance criteria.

In the absence of such a system and to select the next agent for translation, the Consortium has developed aTreatment Strategy Selection Committee, and this committee selected the most promising 5 agents for potential trans-lation. The results show that the preclinical work on these 5 agents has left numerous gaps in knowledge about theirpreclinical performance and conrm the need for signicant changes in preclinical neuroprotection/pharmacotherapytrials in SCI. A recommendation is made for the development and validation of a preclinical scoring system involvingworldwide experts in preclinical and clinical SCI.(http://thejns.org/doi/abs/10.3171/2012.5.AOSPINE12116)

KEY WORDS • spinal cord injury • neuroprotection • pharmacotherapy

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Abbreviations used in this paper: BBB = Basso-Beattie-Bres-nahan; MABP = mean arterial blood pressure; MDA = malondial-dehyde; MgSO4 = magnesium sulfate; NACTN = North AmericanClinical Trials Network; PEG = polyethylene glycol; SCI = spinalcord injury; SSEP = somatosensory evoked potential.


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158 J Neurosurg: Spine / Volume 17 / September 2012

have already been many comprehensive reviews of neu-roprotective/pharmacological agents for SCI,6,71,72 and thepresent report will not duplicate these reviews, but ratheris aimed at providing a focused analysis of the eld ofneuroprotection/pharmacotherapy for SCI for the pur-pose of ascertaining the “translational readiness” of a se-

lection of key promising agents.The present review is focused on neuroprotection

afforded by pharmacotherapy, and so omits other neu-roprotective strategies such as hypothermia. The reviewhas 2 objectives: 1) to identify, describe, and discuss thestrengths and weaknesses of the existing preclinical grad-ing systems or recommended criteria (that is, translationalcriteria) for determining whether a given pharmacologi-cal therapy should be translated from the laboratory intoclinical trials; and 2) using the information gained in therst objective as a guide to identify, describe, and sum-marize the characteristics of preclinical trials that evalu-ate 5 neuroprotective agents that the NACTN has deemedto be of current interest and that we have selected because

of their actual or potential for translation as neuroprotec-tion for SCI. These include riluzole, glyburide, MgSO4 (with and without PEG), nimodipine, and minocycline.The preclinical studies of these agents will be discussedand the results put into the context of the translational cri-teria summarized from the rst objective. It is of interestthat a recent publication that scored translational readi-ness of 12 agents for SCI trials included 3 selected by thecurrent authors.40

Methods

Electronic Literature Database

A systematic search was conducted in PubMed forliterature published from 1966 through November 2011.Details of the search may be found in Tables 1 and 2. Re-sults were limited to articles with abstracts published inthe English language. Reference lists of key articles werealso systematically checked.

For our rst objective (Table 3), articles were identi-ed that addressed issues related to the translational read-iness of SCI pharmacological therapies for clinical trials.The primary focus was to identify articles that proposedspecic grading criteria for studies on pharmacologicaltreatment of SCI. Unfortunately, there was a lack of litera-ture on this topic; therefore, the search was expanded toinclude those articles with a primary focus on evaluatingcriteria used to translate a pharmacological therapy frompreclinical to clinical trials (that is, “translational crite-ria”). These criteria include experimental injury models,the timing of therapy, evidence of benecial effects oftherapy, safety and toxicity of therapy, reproducibility/replication and publication of study results, and miscel-laneous issues. Articles were excluded by title or abstractif it was clear that the primary focus was not relevant toSCI translation. Other exclusions included abstracts, let-ters, white papers, and studies not written in English.

For the second objective (Table 4), preclinical stud-

TABLE 1: Search strategy for Key Question 1: grading systems/criteria for translating pharmacological or cell-based SCI therapy from

laboratory to clinical studies*

Search

No. Search Term No. of Articles

1 “Spinal Cord Injuries” [Majr] OR “spinal cord injury” OR “Spinal Cord Injuries/therapy*” [MeSH] 23125

2 “Biomedical Research*” OR “Biomedical Research/methods” [MeSH] OR “Biomedical Research/

trends” [MeSH] OR “Clinical Trials as Topic*” [MeSH] OR “Dif fusion of Innovation*” [MeSH] OR

“Drug Evaluation, Preclinical/methods” [MeSH] OR “Drug Evaluation, Preclinical/standards”

[MeSH] OR “Drug Evaluation, Preclinical/trends” [MeSH] OR “grading system” [ti/abs] OR

“Guidelines as Topic” [MeSH] OR “Translational research” [MeSH] OR “Translational research/

methods” [MeSH] OR “Translational research/standards” [MeSH] OR “Translational research/

trends” [MeSH]

240732

3 1 AND 2 563

4 3 NOT (neoplasm OR cancer OR coronary OR “comparative study” OR “comparative studies” OR

“cross-sectional studies” OR “cross-sectional study” OR “prospective studies” OR “prospective

study” OR “multicenter studies” OR “multicenter study” OR liver OR renal OR urinary OR

disasters OR malnutrition OR “wound*” OR “retrospective studies” OR “retrospective study” OR

“multiple sclerosis” OR “pilot projects” OR “pilot study” OR “adaptation, psychological” OR“grief” OR dogs OR canine* OR “social behavior” OR “pressure ulcer” OR “muscle contraction”

OR “controlled study” OR “controlled studies” OR “randomized controlled trial” OR “randomized

controlled trials” OR “spinal fusion” OR “follow-up study” OR “follow-up studies” OR cats)

234

246 articles reviewed at ti/abs level (234

from PubMed search + 12 articles

identied in hand-searching relevant

bibliographies)

Include at ti/abs review: 26

Include at full-text review: 4

* PubMed search date: 11/16/2011; search limits: English, only items with abstracts. Abbreviation: ti/abs = title/abstract.


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Translational potential of pharmacotherapy for SCI

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ies were identied in which the neuroprotective effects ofriluzole, glyburide, MgSO4, nimodipine, or minocyclinewere evaluated in animal models of SCI (including bluntforce trauma, penetrating trauma, or ischemia). Studieswere excluded by title or abstract if they evaluated fewerthan 5 animals, if the pharmacological agent was deliv-ered prior to SCI, or if they were not explicitly testingsome aspect of the neuroprotective effect of the agent.Studies that clearly indicated that the pharmacologicalagent was being tested in non-SCI models, in models ofinfection- or tumor-based SCI, ex vivo studies, or in vi-tro studies were also excluded. Full-text articles of theremaining studies were obtained and reviewed for inclu-sion. Furthermore, articles that did not contain a controlgroup (that is, SCI plus saline, vehicle, or no treatment)were excluded. Other exclusions included preclinical tri-als without outcome data, studies of human subjects, un-

published data, technique papers, reviews, editorials, andstudies not written in English.

Data Extraction

Each retrieved citation was assessed by 2 reviewers

working independently (R.H. and A.R.). Most articleswere excluded on the basis of information provided bythe title or abstract. Full-text versions of all citations thatappeared to be appropriate, including those that couldnot be excluded unequivocally on the basis of the titleand abstract, were then assessed by the 2 reviewers. Anydisagreement between them was resolved by consensus.The following information was extracted for Objective1: the type of therapy addressed, the basis of the crite-ria, scoring, components of criteria or preclinical studycharacteristics, or issues discussed. For Objective 2 thefollowing data were extracted from the preclinical stud-

TABLE 2: Search strategy for Key Question 2: preclinical studies*

Agent

Search

No. Search Term No. of Articles

riluzole

1 “Spinal Cord Injuries”[Majr] OR “spinal cord injury” OR “Spinal Cord Injuries/therapy*”[MeSH] 231542 “Riluzole” OR “riluzole”[MeSH] 772

3 1 AND 2 27



glyburide

1 “Spinal Cord Injuries”[Majr] OR “spinal cord injury” OR “Spinal Cord Injuries/therapy*”[MeSH] 23154

2 “Glyburide” OR “glibenclamide” OR “Glyburide”[MeSH] 7205

3 1 AND 2 5



MgSO4


2 “magnesium” OR “magnesium sulfate” OR “magnesium sulfate”[MeSH] 546513 1 AND 2 71



nimodipine


2 “nimodipine” OR “nimodipine”[MeSH] 2991

3 1 AND 2 30



minocycline


2 “minocycline” OR “minocycline”[MeSH] 3557

3 1 AND 2 42



* Riluzole: search date, 11/28/2011; search database, PubMed; limits, English—only items with abstracts. Glyburide: search date, 11/28/2011; search

database, PubMed; limits, English—only items with abstracts. MgSO 4: search date, 11/28/2011; search database, PubMed; limits, English—only items

with abstracts. Nimodipine: search date, 12/5/2011; search database, PubMed; limits, English—only items with abstracts. Minocycline: search date,

12/5/2011; search database, PubMed; limits, English—only items with abstracts.


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ies: animal model, injury model, experimental groups,timing of therapy, dosage(s) used, route of intervention,randomization of animals to treatment groups, blindedor independent assessment of outcomes, and reporting ofresults for all animals. Data on the effectiveness of thetherapy (including pathology of the lesion, biochemicalstudies, electrophysiological studies, motor function, sen-sory function, other neurological function, neuropathicpain, and spasticity) and the safety of the therapy, includ-

ing toxicology and adverse events, were also extracted.

Data Analysis

Data for each key question were abstracted in tables.For the rst objective, grading criteria and issues relatedto translational research in SCI, such as animal/injurymodel and efcacy of a therapy were summarized, andstrengths and weaknesses of the group of articles werediscussed. This information was then used to proposecomponents that should be considered when evaluatingwhether a given pharmacological therapy is ready forclinical translation. For the second objective, the preclini-cal studies were summarized and discussed in terms ofthe readiness of the specic agent for clinical transla-

tion. It was hypothesized that this analysis will serve asa prototype methodology to evaluate which characteris-tics best determine whether a therapy is ready for clinicaltranslation and to suggest additional criteria that may beincluded in such an evaluation process.

Results

Objective 1: Review of Criteria for Determining theTranslational Readiness of a Pharmacological TherapyFrom the Laboratory Into Clinical Trials

We identied 246 articles from the literature search

that addressed readiness of SCI pharmacological thera-pies for clinical translation. However, only 26 articleswere judged suitable for full-text review, 22 of whichwere excluded after full-text review for the following rea-sons: 19 articles did not specically address translationresearch of SCI pharmacological therapies, 1 article wasan update of one of the included articles, and 2 articlesaddressed only cell-based therapies. Therefore, only 4 ar-ticles met the inclusion criteria, and only 1 of the 4 (Kwon

et al.41) proposed a scoring system. Although the other 3discussed issues related to the translation of pharmaco-logical therapies in patients with SCI or stroke,3,13,15 theydid not include a scoring system or discuss the relativeimportance of the issues. The grading system proposedby Kwon et al.41 assigns a total score based on the transla-tional potential of a specic therapy, where a higher scoreindicates a potentially more promising experimentaltreatment for a clinical trial. A summary of the ndingsis presented in Table 5.

Animal/Injury Model(s). All 4 articles addressed theissue of the animal species and injury models used instudies of pharmacological therapies. The grading scalein Kwon et al.41 (Table 6) assigned the highest number of

points to treatments in studies using primate and large-animal models (for example: dog, cat, sheep) and fewerpoints for small rodents. Other authors also placed im-portance on the use of research in larger animals,13,15 withAnderson et al.3 proposing that more invasive or higher-risk treatments be tested in the large-animal models. Sev-eral authors stressed that treatments should be deemedeffective in several animal models13,15 before moving toclinical trials, with a lesion in which the volume, location,and origin were representative of the type of SCIs thatoccur in humans.15 Kwon et al.’s grading scale assignedthe highest points to treatments in studies using cervical

TABLE 3: Inclusion and exclusion criteria for Objective 1: translational readiness for SCI

Study Component Inclusion Exclusion

Study design Articles that provide grading criteria to evaluate quality of pre-

clinical trials for clinical translation

Articles in which primary focus is on translational researchgrading criteria, components, or issues

Articles in which primary focus is not on translational research

grading criteria, components, or issues

Articles that don’t specically address SCI translational research issues

Articles in which primary focus is on addressing issues in clinical

research

Intervention Pharmacological agents being evaluated for improving outcomes

following SCI

Pharmacological agents being evaluated for improving outcomes

in other disease models

Hypothermia & other physical modalities

Bioengineered scaffolds

Preclinical study

characteristics

of interest

Type of therapy addressed

Basis of criteria

Scoring

Components of criteria or preclinical study characteristics in-

cluding animal/injury models, timing of therapy, evidence of

benecial effects of therapy, safety & toxicity of therapy,

reproducibility/replication & publication of study results, &

miscellaneous issues

Publication Articles written in English in peer-reviewed literature w/ abs

available

Abs, letters

White papers


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contusion and clip compression SCI models, fewer pointsto thoracic contusion and clip compression, and the low-est number of points to cervical or thoracic partial sharptransection SCI models.

Timing of Therapy. All 4 articles discussed the tim-ing of the proposed therapy and its therapeutic efcacy.Kwon et al.41 assigned a higher number of points to treat-ments that demonstrate efcacy after a longer treatmentdelay postinjury. Dobkin15 and Dietrich13 proposed thatthe timing and dose of the treatment replicate that which

a human patient would receive. Evidence of Benecial Effects of Therapy. All 4 ar-

ticles addressed the issue of judging the benecial effectsof a therapy. Kwon et al.41 assigned points to various be-havioral and nonbehavioral outcome measures, includinglocomotor tests, and considered the use of dose-responseanalysis for behavioral and nonbehavioral outcomes inthe grading scale. Dietrich13 asserted that the quantitativemethods of assessing outcomes after treatment should beclinically relevant. Anderson et al.3 suggested that anytherapeutic benets of a treatment should persist for atleast 3 months after injury to ensure that true differences

exist between treated and untreated animals. Dobkin15 advised caution in the interpretation of behavioral out-comes found in rodent SCI models because many behav-iors tested in rodents cannot be extrapolated to a similarresponse in humans.

Reproducibility/Replication and Publication. All arti-cles stressed the need for study results to be peer-reviewedby independent experts,3,13 published in peer-reviewed journals,3,13,41 and independently replicated3,13,15,41 beforetranslation to clinical trials. The grading system proposed

by Kwon et al.41

assigns a higher number of points to treat-ments having a greater number of studies that report ben-ecial effects of the therapy, with negative points assignedto treatments having studies that report negative results ofthe therapy.

Safety/Toxicity. Surprisingly, only 2 articles addressedthe need for safety or toxicity monitoring in preclinicalstudies. Anderson et al.3 proposed that highly invasive orrisky interventions should meet a higher standard of pre-clinical safety and efcacy. They also stressed that animalsshould be monitored for effects such as pain, worsened au-tonomic dysfunction, and spasticity for a time period de-

TABLE 4: Inclusion and exclusion criteria for Objective 2: preclinical studies of the 5 selected agents*

Study Component Inclusion Exclusion

Intervention Selected neuroprotective agents (riluzole, glyburide, MgSO4,

nimodipine, & minocycline) evaluated for improving outcomes

post-SCI

Neuroprotective agents evaluated for improving outcomes w/ Tx

prior to SCI

Neuroprotective agents not explicitly tested for some aspect oftheir neuroprotective effect

Population Animal models of SCI, including (but not limited to) blunt force

trauma SCI, penetrating trauma SCI, ischemic SCI

Animal models of ALS, MS, etc.

In vitro models of SCI

Ex vivo models of SCI

Characteristics of

interest

Study characteristics, including:

animal model

injury model

experimental groups

timing of therapy

dosage(s) used

route of intervention

randomization of animals to Tx groups

blinded or independent assessment of outcomes

whether all animals were accounted for

Effectiveness of therapy, including:

pathology of lesion & surrounding area

biochemical studies

electrophysiology studies

motor & neurological function

neuropathic pain

spasticity

Safety of therapy, including toxicology & adverse events

Study design Comparative preclinical trials published in peer-reviewed jour-

nals

Studies w/ no control group

Studies evaluating


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signed to detect signicant adverse events. Dietrich13 noted

that safety issues, including toxicity, should be consideredat every phase of the study.

Other. Other issues were noted by authors as needingfurther research or discussion. Dobkin15 suggested thatthe laboratory environment experienced by rodents mightconfound the interpretation of experiments and thus thetranslation of a treatment from animal models to humanstudies. This environment includes deprivation of socialinteraction, exercise, and environmental enrichment. Di-etrich13 suggested that knowledge of the basic mecha-nisms by which a therapy works would assist researchersin revising treatment protocols and investigating cause-

and-effect relationships between treatments and observed

outcomes.Strengths and Weaknesses of Existing Grading Cri-

teria. There are various strengths and weaknesses identi-ed in each of the 4 articles that proposed grading criteriaor issues in the translational research of pharmacologicaltherapies for SCI, which are summarized in Table 7; moredetailed information is available in Table 8. Althoughmost of the articles discussed important issues such asanimal and injury models, reproducibility and publica-tion of study results, and evidence of benecial effectsof the therapy, there are some weaknesses. Most of thearticles did not discuss the relative importance of the is-

TABLE 6: Preclinical grading scale, neuroprotective therapies for acute SCI*

Component Items Points

Max

Score

Animal species in which efcacy has

been demonstrated

Models of traumatic SCI

primate large animal

rat

mouse

86

4

2

20

Injury paradigms in which efcacy has

been demonstrated

SCI models

cervical contusion

thoracic contusion

cervica l clip compression

thoracic clip compression

cervica l partial transection, sharp

thoracic partial transection, sharp

6

3

6

3

1

1

20

Time window of efcacy Efcacy demonstrated w/ Tx

delay of ≥12 hrs

delay of ≥4 hrs &


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sues raised.3,13,15 Several articles made little or no mentionof safety or toxicity,15,41 and none of the articles discussedthe importance of randomizing the treatment or havingthe investigators work in a blinded fashion. The gradingsystem of Kwon et al.41 has several additional weaknesses:there was no consideration of the commercial potentialof a given therapy, only scientists or spine surgeon–sci-entists were invited to the focus group meeting that de-cided the weighting factors for each subscale in the grad-ing criteria, and the focus group was composed mainly ofAmerican and Canadian researchers.

Objective 2: Preclinical Characteristics and Outcomes forthe 5 Selected Neuroprotective Agents

The following is a detailed account of the 5 agentsselected for analysis on the basis of their promise of use-fulness for translation to human SCI.

Glyburide

Background.

The management of parenchymal hem-orrhage has been reported to be critical for promotingneurological recovery after SCI.22 Glyburide works pri-marily by blocking sulfonylurea receptor 1 (SUR1)–regu-lated, Ca2+-activated, [ATP]i-sensitive nonspecic cation(NCCa-ATP) channels, which helps mitigate the effects ofsecondary hemorrhage and progressive hemorrhagic ne-crosis following SCI.57,64,65 Glyburide has been approvedby the FDA for the treatment of Type 2 diabetes at a doseof 1.25–20 mg (standard) or 0.75–12 mg (micronized)orally in 1 or 2 divided doses.

Systematic Search for Preclinical Studies. Of the 5studies identied in our literature search, 3 that evaluatedglyburide (glibenclamide) in preclinical studies were se-

lected to undergo full-text review, and all 3 met the inclu-sion criteria.57,64,65

Study Characteristics. Study characteristics are sum-marized in Table 9; detailed information can be foundin Table 10. All 3 studies used rats, and no other specieswere used to test the effects of glyburide. The total num-ber of animals per study ranged from 10 to 54 (1 study didnot report the number), with 3–8 animals per treatmentgroup. A blunt force weight-drop injury model was usedin all 3 studies to injure the cervical spine. All 3 studiescompared glyburide to vehicle/saline following SCI, andSimard et al.64 additionally included a no SCI/no treat-

ment group. Simard et al.65 gave a loading dose of 10 mg/kg glyburide within 15 minutes of SCI, and Popovich etal.57 did the same in 1 of 3 experiments; otherwise, infu-sion began within minutes of the injury. Glyburide wasadministered continuously at a rate of 200 ng/hour viaa subcutaneous osmotic pump in all studies; vehicle/sa-line was given similarly. No studies evaluated the dose-response effects of glyburide. In all 3 studies, blinded in-vestigators evaluated outcomes. Popovich et al.57 random-ized animals to treatment groups, whereas no mention ofrandomization was made by the other 2 studies. Popovichet al. accounted for all 54 animals included in the study,but the other 2 studies did not report whether all animalswere accounted for.

Effectiveness of Therapy. The effect of glyburide onintraspinal hemorrhaging was evaluated by 2 studies. Si-mard et al.64 concluded that glyburide treatment resultedin less hemorrhaging compared with the control groupsat 24 hours, as measured by spectrophotometric analysisof blood in the cord homogenates (p < 0.05 at 6, 12, 18,and 24 hours), as well as by other assays. Popovich et al. 57 reported that glyburide given continuously at a dose of200 ng/hour resulted in no differences in the amounts ofvisible hemorrhaging between treatment groups at up to24 hours postinjury in one experiment. However, in a sec-ond similar experiment, and in a third in which a loadingdose was given after injury and followed with continuousinfusion of lower doses, less hemorrhaging was visiblein the glyburide group compared with the SCI/vehiclegroup at 24 hours. There were slight differences betweenthese experiments, including details of the weight dropinjury, which may have contributed to differences in theseresults. The studies performed by Simard et al. in 2007

and 2010 both suggested that glyburide treatment resultedin less visible hemorrhaging compared with the controlgroups at 24 hours. Lesion size was also shown to be sig-nicantly reduced at 1 week64 and 6 weeks65 postinjuryin animals treated with glyburide compared with vehiclein the 2 studies in which this outcome was evaluated (p< 0.001).

Functional improvements in glyburide-treated ratswere observed in all 3 studies. Two studies reportedimprovements in spontaneous rearing (vertical explora-tion), which was statistically higher in animals treatedwith glyburide compared with vehicle at each time point

TABLE 7: Summary of strengths and weaknesses of studies that address translational readiness of a pharmacotherapy for SCI trials

Strengths Weaknesses

Large range of animals & use of relevant injury models, including randomization

& blinding, & adequate numbers of animals

No consideration of randomization or blinding

Appropriate timing of therapy No discussion of relative impor tance of grading issuesEvidence of benecial effects on the basis of a range of outcome measures No consideration of commercial potential of therapy

Safety/toxicity included in evaluation Only scientists/spine surgeons & mostly Americans &/or Canadians

in focus groups

Reproducibility/replication & detailed publication of study results Little mention of lab environment’s effects on interpretation of study

results

Little mention of knowledge of basic mechanisms by which a therapy

works


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T A B L E 8 : G r a d i n g c r i t e r i a a n d i s s u

e s r e l a t e d t o t r a n s l a t i o n r e s e a r c h o f p h a r m a c o l o g i c a l t h e r a p i e s f o r S C I *

A u t h o r s & Y e a r

T y p e o f T h e r a p y

B a s i s o f C r i t e r i a †

S c o r i n g

C o m p o n e n t s o r I s s u e s

S t r e n g t h s / W e a k n e s s e s

K w o n e t a l . ,

2 0 1 1

( “ g r a d i n g

s y s t e m ” )

N e u r o p r o t e c t i v e

t h e r a p i e s f o r

a c u t e S C I

P e r s p e c t i v e s / o p i n i o n s o f 2 0 0 +

c l i n i c i a n s & s c i e n t i s t s i n

S C I e l d v i a q u e s t i o n n a i r e

F i n a l c r i t e r i a b y c o n s e n s u s o f

2 5 i n v i t e d s c i e n t i c e x p e r t s

& s p i n e s u r g e o n – s c i e n t i s t s

i n m o d i e d D e l p h i e x e r c i s e

0 – 1 0 0 ; e x t e n t

t o w h i c h a

p a r t i c u l a r T

x h a s b e e n

s t u d i e d , w /

h i g h e r

s c o r e i n d i c a t i n g a

g r e a t e r b o d

y o f p e e r -

r e v i e w e d l i t e r a t u r e

P r o v i d e s “ a n o b j e c t i v e m e a s u r e o f t h e ‘ t r a n s l a -

t i o n a l p o t e n t i a l ’ o f a s p e c i c t h e

r a p y b a s e d o n

a s y s t e m a t i c a l l y c o l l e c t e d s e t o f l i t e r a t u r e

s u p p o r t i n g i t s a p p l i c a t i o n i n a c u

t e S C I ”

A n i m a l m o d e l s i n w h i c h e f c a c y h

a s b e e n d e m -

o n s t r a t e d

I n j u r y m o d e l s i n w h i c h e f c a c y h a

s b e e n d e m o n -

s t r a t e d

T i m e w i n d o w o f e f c a c y

D e m o n s t r a t i o n o f “ c l i n i c a l l y m e a n i n g f u l ” e f c a c y

I n d e p e n d e n t r e p r o d u c i b i l i t y / r e p l i c a t i o n

N o c o n s i d e r a t i o n o f c o m m e r c i a l p o t e n t i a l o f

t h e r a p y

F u l l s p e c t r u m o f i n t e r n a t i o n a l

i n p u t s o m e w h a t

l a c k i n g

P r o p o s e d T x c a n p o s s i b l y a c h

i e v e a h i g h e r s c o r e

i n a b s e n c e o f s o m e e l e m e n t s

E x c l u d e d s a f e t y ( d e e m e d t o b

e a r e g u l a t o r y

i s s u e f o r a l l n e u r o p r o t e c t i v e a g e n t s )

O n l y s c i e n t i s t s o r s p i n e s u r g e

o n – s c i e n t i s t s ( P I s )

i n v i t e d t o f o c u s g r o u p m e e t i n g t h a t d e c i d e d

w e i g h t i n g f a c t o r s f o r e a c h s u b s c a l e

Q u a l i t y o f s c i e n t i c p r e c l i n i c a l S C I s t u d i e s n o t

a s s e s s e d

N e e d s t o b e m o d i e d f o r o t h e

r t h e r a p i e s ( c e l l

t r a n s p l a n t a t i o n o r b i o l o g i c a

l t h e r a p i e s )

D o b k i n ,

2 0 0 7

N e u r a l r e p a i r a f t e r

s t r o k e o r S C I

N A

N o n

e

A n i m a l m o d e l s d e s i g n e d t o b e r e l e v a n t t o h u m a n

d i s e a s e

D i f f e r e n c e s b t w n r o d e n t b r a i n / s p i n a l c o r d & h u -

m a n C N S

D i f f e r e n c e s b t w n v a r i o u s r o d e n t m

o d e l s ( r e p l i c a -

t i o n o f s a m e i n j u r y & r e p a i r m o d e l i n d i f f e r e n t

l a b s o f t e n n o t p e r f o r m e d )

R e p r o d u c e k e y r e s u l t s i n > 1 l a b

D i f f e r e n c e s i n i n j u r y i n d u c t i o n

D o s e & t i m i n g o f i n t e r v e n t i o n s

A n a t o m i c a l s i t e & g o a l o f i n t e r v e n t i o n s

C o m b i n a t i o n a l t h e r a p i e s & m u l t i p l e i n t e r v e n t i o n s

L a b e n v i r o n m e n t ( f o r e x a m p l e , a n

i m a l s ’ i s o l a t i o n

o r d e p r i v a t i o n ) , r e h a b i l i t a t i o n , p o s t l e s i o n r e -

o r g a n i z a t i o n

P h y s i o l o g i c a l , h i s t o l o g i c a l , & m o l e

c u l a r m a r k e r s

c a n s e r v e a s s u r r o g a t e s f o r b e h a v i o r a l o u t -

c o m e s , & l i m i t e d r o d e n t b e h a v i o r s a r e t e s t e d

N o i n d i c a t i o n o f r e l a t i v e i m p o r t a n c e o f i s s u e s

L i t t l e m e n t i o n o f s a f e t y , a d v e r s e e v e n t s , o r

t o x i c o l o g y

( c o n t i n u e d )


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T A B L E 8 : G r a d i n g c r i t e r i a a n d i s s u

e s r e l a t e d t o t r a n s l a t i o n r e s e a r c h o f p h a r m a c o l o g i c a l t h e r a p i e s f o r S C I * ( c o n t i n u e d )

A u t h o r s & Y e a r



S c o r i n g


S t r e n g t h s / W e a k n

e s s e s

A n d e r s o n e t

a l . , 2 0 0 5

P h a r m a c o l o g i c a l ,

c e l l - b a s e d ,

&

o t h e r t h e r a p i e s

f o r S C I

B a s e d o n g u i d e l i n e s i s s u e d

b y t h e A S N T R ( s e e R a m e r

e t a l . )

N o n e

A p p r o p r i a t e d a t a s h o u l d b e a c q u i r e d i n a n i m a l s

p r i o r t o h u m a n s t u d i e s , i n c l u d i n g :

I n j u r y m o d e l i n h u m a n s s h o u

l d b e v a l i d a t e d

b y p r e c l i n i c a l m o d e l i n a n i m a l s ( f o r

e x a m p l e , c o n t u s i o n o r i s c h e m i a )

T i m e f r a m e o f T x i n a n i m a l s s h o u l d r e p l i -

c a t e t h a t o f a c u t e o r c h r o n

i c t h e r a p i e s i n

h u m a n s

R i s k i e r i n t e r v e n t i o n s s h o u l d m e e t h i g h e r

s t a n d a r d o f s a f e t y / e f c a c y

T x s h o u l d h a v e d e m o n s t r a b l e & s t a t i s t i c a l l y

s i g n i c a n t b e n e t i n a n i m a l m o d e l s

S t u d y r e s u l t s s h o u l d b e p e e r - r e v i e w e d b y

i n d e p e n d e n t e x p e r t s

O u t c o m e s s h o u l d b e a s s e s s e d a t c e l l u l a r ,

p h y s i o l o g i c a l , & b e h a v i o r a

l l e v e l s i n a n

a p p r o p r i a t e m o d e l o f S C I

P e r s i s t e n c e o f t h e r a p e u t i c b e

n e t s h o u l d

b e s h o w n f o r a t l e a s t 3 m o

s p o s t i n j u r y

E v i d e n c e o f i n d e p e n d e n t r e p

l i c a t i o n s h o u l d

b e p r e s e n t b e f o r e t r a n s l a t i o n

A n i m a l m o d e l s s h o u l d b e m o

n i t o r e d f o r

s a f e t y / a d v e r s e e f f e c t s ( p a i n , w o r s e n e d

a u t o n o m i c d y s f u n c t i o n , s p

a s t i c i t y ) i n s y s -

t e m s a b o v e t h e l e s i o n f o r a t i m e p e r i o d

d e s i g n e d t o d e t e c t s i g n i c

a n t a d v e r s e

e v e n t s

N o i n d i c a t i o n o f r e l a t i v e i m p o r

t a n c e o f i s s u e s

( c o n t i n u e d )


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167

measured, ranging from 1 day to 1 week57 or 6 weeks (p< 0.01).65 Two studies found improvements in inclined-plane test scores in animals treated with glyburide com-pared with vehicle as measured between 1 and 7 days (p <0.05),57,64 and 1 study each reported improvements in ip-silateral paw placement64 and in BBB scores (p < 0.001)57

at 1 day following injury.Safety of Therapy. None of the studies assessed the

safety of glyburide.

Summary of Preclinical Trials of Glyburide. Thus,according to the criteria shown in Tables 5 and 7, thereare numerous deciencies in the translational readinessof this agent.

Magnesium Sulfate

Background. For the treatment of SCI, the primarymechanism of action for MgSO4 appears to be limitinglevels of intracellular calcium by blockage of N -methyl-D-aspartate receptors and of voltage-gated calcium chan-nels, subsequently reducing free radical generation, gluta-

mate release, expression of p53-related proteins, lipid per-oxidation, lactate accumulation, and cell death.24,44,46,78,79,86 Magnesium sulfate is currently indicated in humans forthe immediate control of life-threatening convulsions inthe treatment of severe toxemias (preeclampsia and ec-lampsia) of pregnancy, for the treatment of acute nephri-tis in children, and as a replacement therapy in MgSO4 deciency, especially in acute hypomagnesemia accom-panied by signs of tetany. It is also used to prevent prema-ture contractions in pregnancy and to treat patients withheart attack and asthma. The drug can be administeredintramuscularly or intravenously, and the dose is veryvariable, ranging from 1 to 3 g daily for a maintenancedose for adults, to 10–14 g for severe preeclampsia or ec-

lampsia.Systematic Search for Preclinical Studies. We identi-

ed 71 articles that evaluated MgSO4, of which 14 wereeligible for full-text review. Five studies were excludedafter full-text review for the following reasons: 4 studiesadministered MgSO4 prior to injury, and 1 study did notevaluate MgSO4. Therefore, 9 articles met our inclusioncriteria.14,26,35,36,42,53,66,69,81

Study Characteristics. Study characteristics are sum-marized in Table 11; detailed information can be foundin Table 12. Administration of MgSO4 following SCIwas tested in rats in 8 of the studies14,26,35,36,42,66,69,81 andin rabbits in 1 study (Ozdemir et al.).53 The total numberof animals per study ranged from 30 to 122, with 6–20

animals per treatment group. Most studies evaluated tho-racic SCI: blunt force trauma by weight drop was used in6 studies26,35,36,42,66,81 and clip compression in 2 (Ditor etal.14 and Süzer et al.69); 1 study provided no details on theinjury model.53 In all studies, MgSO4 was administeredsystemically, 5 intraperitoneally26,35,66,69,81 and 4 intrave-nously.14,36,42,53 The dosages of MgSO4 used were 60 mg/kg (2 doses nearly 6 hours apart),42 100 mg/kg,35,53 300 mg/kg,14,69 and 600 mg/kg.26,35,36,66,69,81 Only Kaptanoglu et al.35 and Süzer et al.69 evaluated more than one dose of MgSO4.All but 1 study gave the rst injection of MgSO4 within0–15 minutes postinjury; Süzer et al. administered the T

A B L E 8 : G r a d i n g c r i t e r i a a n d i s s u

e s r e l a t e d t o t r a n s l a t i o n r e s e a r c h o f p h a r m a c o l o g i c a l t h e r a p i e s f o r S C I * ( c o n t i n u e d )

A u t h o r s &

Y e a r



S c o r i n g


S t r e n g t h s / W e a k n

e s s e s

D i e t r i c h ,

2 0 0 3

N e u r o p r o t e c t i v e o r

r e g e n e r a t i v e

t h e r a p i e s f o r

S C I

N R

N o n e

T h e r a p y m u s t w o r k i n s e v e r a l a n i m

a l m o d e l s ,

i n -

c l u d i n g c o n s i d e r a t i o n o f a n i m a l

s p e c i e s , s e x ,

& l a r g e a n i m a l m o d e l s ( n o n h u m

a n p r i m a t e s )

C o m p e l l i n g e v i d e n c e o f b e n e t , i n c l u d i n g t h e d e -

g r e e o f b e n e t , a w i d e t h e r a p e u

t i c w i n d o w ,

d o s i n g , c l i n i c a l l y r e l e v a n t m e t h o

d s o f a s s e s s -

i n g o u t c o m e ,

& i d e a l l y a n u n d e r s t a n d i n g o f

b a s i c m e c h a n i s m s b y w h i c h t h e

t h e r a p y w o r k s

S t u d y i s c l i n i c a l l y r e l e v a n t & r e p l i c a t e d i n a n i n -

d e p e n d e n t l a b

M a j o r n d i n g s a r e p u b l i s h e d i n p e e r - r e v i e w e d

j o u r n a l s

S a f e t y i s s u e s , i n c l u d i n g t o x i c i t y , a r e a d d r e s s e d a t

e v e r y t e s t i n g p h a s e

C o n s i d e r a t i o n o f a c u t e , s u b a c u t e ,

& c h r o n i c i n -

j u r y s e t t i n g s

N o i n d i c a t i o n o f r e l a t i v e i m p o r t a n c e o f c o m p o -

n e n t s

* A S N T R = A m e r i c a n S o c i e t y f o r N e u

r a l T r a n s p l a n t a t i o n a n d R e p a i r ; P I = p r i n c i p a l i n

v e s t i g a t o r .

† T h e m e t h o d o r s o u r c e u s e d t o p r o p

o s e c r i t e r i a o r i s s u e s i n t r a n s l a t i n g p h a r m a c o l o

g i c a l t h e r a p i e s f r o m p r e c l i n i c a l t o c l i n i c a l s t u d i e

s .


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drug at 1 hour. Wiseman et al.81 conducted 2 experiments,one in which MgSO4 was given at 10 minutes, and anotherin which animals received an injection at either 8, 12, or24 hours. In 2 studies injections were given at 15 minutesand again at 6 hours: in Ditor et al.14 at 300 mg/kg/injectionand Kwon et al.42 at 60 mg/kg/injection. All but 1 study53 clearly stated that animals were randomized to treatmentgroups, and that assessment was done in a blinded manner;

all but 2 studies53,66 used blinded assessment of treatmentoutcomes. Five studies accounted for 100% of the ani-mals;14,26,35,42,69 1 study accounted for 100% of the animalsin the rst experiment, but did not account for the animalsin the second experiment;81 3 studies did not account for allincluded animals.36,53,66

Effectiveness of Therapy.

Lesion size was assessedby 2 studies. Kwon et al.42 found that 6 weeks after SCI,intravenous administration of MgSO4 (2 doses of 60 mg/kg, one at 15 minutes, and the other at 6 hours) reducedlesion volumes by 33% compared with rats that receivedsaline alone (p = 0.03). When MgSO4 was given in PEG,

lesion volumes were reduced even more, by 51% com-pared with saline controls (p = 0.0012) (PEG alone re-duced lesion volumes by 20% compared with salinecontrols). The delivery of MgSO4 in PEG signicantlyreduced lesion size compared with administration ofMgSO4 alone (p = 0.0386). In contrast, Wiseman et al.

81 reported no differences in lesion lengths between treat-ment groups measured 24 hours postinjury, but noted that

the small number of specimens and the large variabilityin the control group samples may have affected the re-sults. The authors reported signicantly improved whitematter sparing following MgSO4 (and methylpredniso-lone + MgSO4) treatment compared with saline alone: thepercentage of myelin preservation was 20.2% in the sa-line group compared with 32.3% (p = 0.002) for MgSO4,30.3% (p = 0.006) for methylprednisolone, and 42.3% (p= 0.0007) for the 2 drugs combined. In this study, ratswere treated with 600 mg/kg MgSO4 (and/or 30 mg/kgmethylprednisolone) 10 minutes after injury.

The effects of MgSO4 on neuronal apoptosis follow-

TABLE 9: Summary of glyburide preclinical study characteristics*

Characteristic Popovich et al., 2012 Simard et al., 2007 Simard et al., 2010

animal model: rats

no. of animals 54 NR 10

no./group 6–8 3–5 5injury model: blunt force (wt drop)

cervical X X X

timing of intervention postinjury

2–3 min w/ cont infusion X X

15 min w/ cont infusion for 7 days X

route of intervention: osmotic pumps placed caudal to injury

in subcutaneous pocket X X X

IP

dosage

200 ng/hr cont infusion until planned death X X

initial dose of 10 μ g/kg; cont infusion of 200 ng/hr for 7 days X

>1 dose evaluated?

yes

no X X X

control groups

SCI (saline, DMSO) X X X

no SCI X

independent or blind assessment

yes X X X

no

NR

animals randomized to Tx groups

yes X

no

NR X X

% animals accounted for 100% (54 of 54) NR NR

* Cont = continuous; DMSO = dimethyl sulfoxide; IP = intraperitoneal.


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ing SCI were examined by Solaroglu et al.66 by measuringcaspase-3 activity. A single dose of MgSO4 (600 mg/kg)was given immediately following weight drop injury, andanimals were killed at 24 hours. Compared with no treat-ment or vehicle alone, MgSO4 reduced caspase-3 activitylevels (p < 0.05) in experimental animals, as did meth-

ylprednisolone. Of note, methylprednisolone treatmentresulted in a greater reduction in caspase-3 activationcompared with MgSO4 (p < 0.05). Caspase-3 activity wasmeasured in 1-cm samples of spinal cord tissue homog-enates taken around the injury site. The results suggestthat MgSO4 may have antiapoptotic effects in the rst 24hours following SCI.

Kaptanoglu et al.35 reported the ultrastructural nd-ings in a 1-mm cross-section of the spinal cord obtainedat the trauma site after 2 different doses of MgSO 4 (100and 600 mg/kg) given immediately after weight drop SCIin rats killed at 24 hours. The higher dose of MgSO4 sig-nicantly improved the general neural score, which as-sessed whole subcellular changes, compared with the no

treatment or saline controls (p < 0.001); in fact, the scoresfrom the animals that received this dose were statisticallysimilar to those in the no injury control group. This ef-fect was not seen with the lower dose of MgSO4 (100 mg/kg). Similar results were seen for the higher but not lowerdose of MgSO4 for measurements of intracytoplasmicedema, nuclear protection, and axon myelin. Both dosesof MgSO4 improved the axonal score (a measure of axo-nal injury) compared with the no treatment control (p <0.05), and the improvements were statistically similar tothe no injury control group.

Spinal cord lactate and/or MDA levels were assessedin 2 studies. Lactate accumulation and MDA formationoccur following neural injury. Ozdemir et al.53 found that

rabbits treated with 100 mg/kg MgSO4 5 minutes afterSCI had signicantly lower lactate and MDA levels thananimals treated with saline. Furthermore, these levels werestatistically similar to those seen in the no injury controlanimals. Süzer et al.69 reported similarly decreased MDAlevels 24 hours postinjury in rats treated with a higher dose(600 mg/kg) but not a lower dose (300 mg/kg) of MgSO4 1 hour after SCI.

Gok et al.26 reported that inltration of neutrophilsinto the spinal cord region, as evaluated by myeloperoxi-dase activity of spinal cord homogenates, was signicant-ly reduced in animals treated with MgSO4 (or methyl-prednisolone) compared with saline (p < 0.05). Treatmentwas given immediately after trauma; the time at whichthe tissue samples were collected was not reported.

Vascular permeability was assessed at 2 and 24 hoursfollowing SCI in rats treated with 600 mg/kg MgSO 4 im-mediately after trauma by Kaptanoglu and colleagues.36 The authors evaluated the extent of blood–spinal cordbarrier damage and the increase in microvascular per-meability by using a 10-minute perfusion of Evans bluedye. Animals treated with MgSO4 had lower Evans bluecontent and thus lower vascular permeability than thetrauma group at both 2 and 24 hours, although the Evansblue content was still higher than in the no trauma controlanimals. Furthermore, Evans blue content increased withtime in the trauma group who received no MgSO4. These

T A B L E 1 0 : C h a r a c t e r i s t i c s o f S C I a

n i m a l s t u d i e s u s i n g g l y b u r i d e ( g l i b e n c l a m i d e ) * ( c o n t i n u e d )

A u t h o r s &

Y e a r

A n i m a l & I n j u r y M o d e l s

E x p e r i m e n t a l

G r o u p s

I n

t e r v e n t i o n D e t a i l s

R e p o r t e d O u t c o m e s

C o m m e n t s

S i m a r d e t a l . ,

2 0 1 0

A n i m a l m o d e l :

R a t s

N = 1 0

S C I m o d e l :

R a t s h a d a u n i l a t c e r v i c a l

i n j u r y

S C I + g l i b e n c l a m i d e

( n = 5 )

S C I + s a l i n e / D M S O

( n = 5 )

R o u t e : s u b c u t a

n e o u s i n j

D o s a g e & t i m i n

g :

W / i n 1 5 m i n o f

S C I , a l o a d i n g d o s e o f g l i b e n -

c l a m i d e ( 1 0

µ g / k g ) w a s g i v e n I P

C o n t i n f u s i o n o

f g l i b e n c l a m i d e ( 2 0 0 n g / h r

s u b c u t a n e o u s l y f o r 7 d a y s )

A n i m a l s k i l l e d :

N R

G l i b e n c l a m i d e T x i n r a t s r e s u l t e d i n s i g n i c a n t l y ( p < 0 . 0

1 )

b e t t e r p e r f o r m a n c e i n s p o n t a n e o u s r e a r i n g d u r i n g 6 w k s

o f o b s e r v a t i o n c o m p a r e d t o v e h i c l e - t r e a t e d c o n t r o l s

G l i b e n c l a m i d e T x i n r a t s r e s u l t e d i n a 3 – 4 ×

r e d u c t i o n i n

l e s i o n v o l c

o m p a r e d w / v e h i c l e - t r e a t e d r a t s ( p < 0 . 0

0 1 )

G l i b e n c l a m i d e T x i n r a t s r e s u l t e d i n a b s e n c e o f c y s t f o r m a -

t i o n & s p i n a l c o r d c a v i t a t i o n ,

b o t h o f w h i c h w e r e p r e s e n t

i n v e h i c l e - t r e a t e d r a t s

S a f e t y / A d v e r s e E v e n t s / T o x i c o l o g y : N R

* B B B = B a s s o - B e a t t i e - B r e s n a h a n m

o t o r s c o r e ; E x p t = e x p e r i m e n t ; i n j = i n j e c t i o n ; N C C a - A T P = C a 2 + - a c t i v a t e d ,

[ A T P ] i - s e n s i t i v e n o n s

p e c i c c a t i o n ; O S U = O h i o S t a t e U n i v e r s i t y ; S U R 1 = s u l f o n y l u r e a

r e c e p t o r 1 ; U M = U n i v e r s i t y o f M a r y l a n d .


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171

T A B L E 1 1 : S u m m a r y o f M g S O

4 p r e c l i

n i c a l s t u d y c h a r a c t e r i s t i c s *

C h a r a c t e r i s t i c

D i t o r e t a l . ,

2 0 0 7

G o k e t a l . ,

2 0 0 7

K a p t a n o g l u e t a

l . ,

2 0 0 3 3 5

K a p t a n o g l u e t

a l . , 2 0 0 3 3 6

K w o n e t a l . ,

2 0 0 9

O z d e m

i r e t a l . ,

2 0 0 5

S o l a r o g l u e t

a l . , 2 0 0 5

S ü z e r e t a l . ,

1 9 9 9

W

i s e m a n e t a l . , 2 0 0 9

a n i m a l m o d e l ( n o . / g r o u p )

r a t s

3

2 ( 6 – 1 1 )

3 5 ( 7 )

5 0 ( 1 0 )

4 2 ( 6 )

5 0 ( 1 0 )

4 0 ( 8 )

3 0 ( 1 0 )

1 2 2 ( 1 3 – 2 0 )

r a b b i t s

3 0

( 1 0 )

i n j u r y m o d e l

b l u n t f o r c e ( w t d r o p ; t h o r a c i

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