Minocycline Reduces Traumatic Brain Injury-mediated Caspase1 Activation, Tissue Damage, and...

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Minocycline Reduces Traumatic Brain Injury-mediatedCaspase-1 Activation, Tissue Damage, and

Neurological Dysfunction

Rene O. Sanchez Mejia, B.A., Victor O. Ona, M.D.,Mingwei Li, M.D., Robert M. Friedlander, M.D.

Neuroapoptosis Laboratory and Department of Neurosurgery, Brigham and Women’sHospital, Harvard Medical School, Boston, Massachusetts

OBJECTIVE: Caspase-1 plays an important functional role mediating neuronal cell death and dysfunction afterexperimental traumatic brain injury (TBI) in mice. Minocycline, a derivative of the antibiotic tetracycline, inhibitscaspase-1 expression. This study investigates whether minocycline can ameliorate TBI-mediated injury in mice.

METHODS: Brains from mice subjected to traumatic brain injury underwent immunohistochemical analyses forcaspase-1, caspase-3, and a neuronal specific marker (NeuN). Minocycline- and saline-treated mice subjected totraumatic brain injury were compared with respect to neurological function, lesion volume, and interleukin-1bproduction.

RESULTS: Immunohistochemical analysis revealed that activated caspase-1 and caspase-3 are present in neurons 24hours after TBI. Intraperitoneal administration of minocycline 12 hours before or 30 minutes after TBI in miceresulted in improved neurological function when compared with mice given saline control, as assessed byRotarod performance 1 to 4 days after TBI. The lesion volume, assessed 4 days after trauma, was significantlydecreased in mice treated with minocycline before or after trauma when compared with saline-treated mice.Caspase-1 activity, quantified by measuring mature interleukin-1b production by enzyme-linked immunosorbentassay, was considerably increased in mice that underwent TBI, and this increase was significantly diminished inminocycline-treated mice.

CONCLUSION: We show for the first time that caspase-1 and caspase-3 activities localize specifically withinneurons after experimental brain trauma. Further, these results indicate that minocycline is an effective phar-macological agent for reducing tissue injury and neurological deficits that result from experimental TBI, likelythrough a caspase-1-dependent mechanism. These results provide an experimental rationale for the evaluation ofminocycline in human trauma patients. (Neurosurgery 48:1393–1401, 2001)

Key words: Apoptosis, Caspase, Interleukin-1b, Minocycline, Traumatic brain injury

Traumatic brain injury (TBI) is the leading cause of deathand disability in young people, resulting in 52,000deaths and 80,000 cases of permanent severe traumatic

brain injury per year in the United States (17, 44). Despite thehigh societal cost and the extensive basic and clinical researchin TBI, there are presently no proven effective pharmacolog-ical interventions apart from those utilized for resuscitation,decreasing intracranial pressure, and maintaining cerebralblood flow (17). TBI consist of a primary injury from a directimpact that results in direct tissue injury and necrotic celldeath. In the ensuing days, a subsequent secondary injuryevolves, characterized by increased intracranial pressure anddecreased cerebral blood flow. Decreased cerebral blood flow

can lead to ischemic events that exacerbate the initial insultthrough additional neuronal cell loss. Many molecular andcellular mechanisms have been proposed to underlie neuronalcell death resulting from secondary injury. Several pharma-cological interventions—including free radical scavengers,N-methyl-d-aspartate antagonists, and calcium channel block-ers—have been developed based on these underlying mech-anisms, although studies have shown no significant improve-ment in clinical outcomes (4). More recently, we and othershave demonstrated that apoptotic pathways are activated andcontribute to neuronal cell loss and neurological dysfunctionafter TBI (6, 9, 12, 31, 36, 45). Apoptotic cell death has alsobeen demonstrated in humans after cerebral contusions, sup-

1393Neurosurgery, Vol. 48, No. 6, June 2001

porting its relevance as a therapeutic target (30). New thera-pies are needed and these newly implicated mechanisms ofcell death may provide good targets for such new agents.

The caspase family, the Bcl-2 family, and the apoptoticprotease-activating factor (Apaf-1) regulate mammalian apo-ptotic cell death (1, 49, 51). Caspase-1, a cysteine protease andthe first member of the caspase family identified, is a mam-malian homolog of the Caenorhabditis elegans death gene prod-uct CED-3, which together with CED-4 (a homolog of Apaf-1)mediates apoptosis in the nematode (48, 50). Caspase-1, alsoknown as the interleukin-1b (IL-1b)-converting enzyme (ICE)for its ability to catalyze the conversion of pro-IL-1b intomature IL-1b, has an important role mediating apoptosis in avariety of experimental paradigms (5, 16, 21, 28, 29, 41, 43, 52).Caspase-1 activation, as demonstrated by the detection ofmature IL-1b, has been identified during apoptosis both invitro as well as in vivo (7, 12, 15, 18, 19, 21, 25, 26, 28, 32, 34,52). We have previously demonstrated that a transgenicmouse expressing a dominant negative inhibitor of caspase-1has decreased ischemia-induced cerebral infarct and de-creased traumatic brain lesion size. Reduced tissue injury andimproved neurological function correlate with decreased ma-ture IL-1b production (12, 15, 25). Several reports have re-vealed that IL-1b itself has a functional role in apoptosis (16,43). We have further demonstrated a role of caspase-1 medi-ated neurodegeneration in animal models of Huntington’sdisease and ALS as well as acute neurological insults such ascerebral ischemia, brain trauma, and spinal cord injury (11, 12,14, 15, 18, 19, 25, 26, 32, 35). The above evidence implicatescaspase-1 as an important mediator of cell death in a varietyof neurological conditions. Caspase-3, an effector caspasedownstream of caspase-1, has also been implicated in neuro-nal cell death due to acute and chronic neurological insults (7,25, 26, 38, 45). Recently others have reported that caspase-3-like proteases are activated and contribute to neurologicaldysfunction after TBI (8, 45). These studies found thatcaspase-3 expression and activity was increased, and can bedetected immunohistochemically in apoptotic cells after ex-perimental TBI. Furthermore, administration of a caspase-3peptide inhibitor decreased apoptosis and improved neuro-logical function after TBI.

Tetracycline and its derivatives doxycycline and minocy-cline, in addition to having antibiotic properties, were re-cently found to have anti-inflammatory and anti-apoptoticproperties (2, 7, 33, 46, 47). In experimental cerebral ischemiathese actions are at least partially due to down-regulation ofcaspase-1 and the inducible form of nitric oxide synthase(iNOS) (46, 47). We demonstrated that minocycline slowsdisease progression in a mouse model of Huntington’s dis-ease, suggesting it could have beneficial effects in chronicneurodegenerative diseases as well (7). The exact mechanismof action for minocycline in preventing inflammation andneuronal cell death in these mouse models is not completelyunderstood but it is likely due to its ability to decrease the ex-pression of caspase-1, caspase-3, iNOS, and cyclooxygenase-2(COX-2) (2, 7, 46, 47). Minocycline is an attractive pharmaco-logical agent because of its oral bioavailability, ability to cross

the blood-brain barrier, and its extensive use in humans for avariety of acute and chronic conditions.

Since caspase-1 and caspase-3 have been demonstrated toplay a functional role in TBI, and minocycline modulates theiractivity, we investigated whether minocycline might have aneffect in ameliorating injury after brain trauma in mice. In thisstudy, we first demonstrate that both active caspase-1 andcaspase-3 are present within neurons in traumatic brain tis-sue. We then show that treatment with minocycline before orafter TBI results in improved motor function, as assessed byRotarod performance, compared with saline-treated animals.Furthermore we demonstrate that minocycline reduces TBI-mediated tissue injury as well as caspase-1 activity. The lackof effective therapies for TBI, the ability of minocycline toameliorate neurological deficits in experimental TBI, and itsextensive use in humans make this a promising agent thatmerits testing in trauma patients.

MATERIALS AND METHODS

Traumatic brain injury

Brain trauma experiments and lesion quantification wereperformed essentially as previously described (6, 12). Sponta-neously breathing adult C57BL/6 mice (Jackson Laboratories,Bar Harbor, ME) were anesthetized with isoflurane in 70%N2O and 30% O2. Mice were positioned in a stereotactic frameand underwent an atraumatic craniotomy removing the rightparietal bone posterior to the coronal, lateral to the sagittal,and anterior to the lambdoid suture. Laterally, the craniotomywas extended to the temporalis muscle insertion. A piston 3mm in diameter with an excursion of 3 mm was positionedover the craniotomy window. A 20 g weight was droppedfrom a height of 150 mm inside a cylinder onto the piston(final speed v 5 1.70 m/s). Temperature was maintained atapproximately 37°C during the procedure and the following24 hours. An investigator naive to the animal identity per-formed the surgical procedure and analyses. The trauma pro-tocol was approved by the Harvard Medical School AnimalCare Committee.

Immunohistochemical staining

Mice were killed 24 hours after TBI and perfused with 4%paraformaldehyde. The brains were removed and frozen inchilled isopentane after cryoprotection in 30% sucrose. Frozensections (12 mm) were prepared and immunohistochemicalanalysis was performed as previously described (26). Acti-vated caspase-1 and caspase-3 was detected immunohisto-chemically using specific antibodies against caspase-1 (pro-vided by J. Yuan, Harvard Medical School, Boston, MA) andcaspase-3 (provided by A. Srinivasan, Idun Pharmaceuticals,La Jolla, CA). The anti-NeuN antibody (Chemicon, Temecula,CA) was used as a neuron-specific marker.

Minocycline treatment

Minocycline HCl (Sigma, St. Louis, MO) was administeredunder two different protocols using different dosages and

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Neurosurgery, Vol. 48, No. 6, June 2001

schedules in animals randomly assigned to control or treat-ment groups.

Pretreatment protocolA dose of 45 mg/kg body weight was injected intraperito-

neally 12 hours before TBI. This dose was selected based ondosages reported to have an effect in an experimental isch-emia model (46, 47). Beginning 30 minutes after trauma, theanimals received minocycline injections every 12 hours, at adose of 90 mg/kg for the first 24 hours after trauma and then45 mg/kg thereafter until the mice were killed. Controls re-ceived equivolume saline.

Posttreatment protocolMice received no treatment before trauma and began the

same treatment protocol 30 minutes after trauma as above.Control groups were treated with equivolume saline underthe same schedule.

Rotarod test

Motor performance was quantified daily using a Rotarod(Columbus Instruments, Columbus, OH) 1 to 4 days aftertrauma. Mice were placed on the rotating rod at 25 rpm andthe time until drop off was automatically registered. If themouse remained on the rod for 10 minutes the test wascompleted and scored 10 minutes.

Lesion size measurement

Four days after trauma, brains were removed and sectionedinto cryostat sections (12 mm) at 0.5 mm intervals from 0.5 mmto 3.0 mm posterior to the bregma and stained with hematox-ylin and eosin. The images of the stained specimens were

captured by a digital photo camera (Sony DKC-5000) andanalyzed by Scion Image (NIH image 6.1 system) for morpho-metric measurement. The total lesion volume was determinedby integrating the volumes at each coronal section interval aspreviously reported (12). A blind investigator performed thelesion volume analyses.

Mature IL-1b determination

Mature IL-1b quantification was performed as previouslydescribed using an enzyme-linked immunosorbent assay kit(R&D Systems, Minneapolis, MN) (19). Brains were removed4 hours after trauma or sham operation, and each hemispherewithout 2 mm from frontal and occipital poles was homoge-nized in lysis buffer. Sham-operated mice were craniecto-mized but not traumatized.

Data analysis

Data are presented as means 6 standard error of the mean.Statistical comparisons were made by Student’s t test. Forcomparison of Rotarod performance two-way analysis of vari-ance followed by Tukey post hoc tests were applied. P , 0.05was considered statistically significant.

RESULTS

Activated caspase-1 and caspase-3 in neurons after TBI

Because caspase-1 and caspase-3 have been implicated inthe pathogenesis of TBI we sought to demonstrate for the firsttime that indeed active caspase-1 (Fig. 1) and caspase-3 (Fig. 2)are present in neurons in the traumatic area. Others havedemonstrated that after experimental TBI caspase-3 colocal-izes to TUNEL-positive apoptotic cells that morphologically

FIGURE 1. Activation ofcaspase-1 after TBI.Brain sections of sham-operated mice (A–D).Brain sections 24 hoursafter TBI (E–H) at lowmagnification. Brain sec-tions 24 hours after TBI(I–L) at high magnifica-tion. Caspase-1 stainingis evident in the neuro-nal cytoplasm andnucleus. Size bars for(A–D) and (E–H) repre-sent 20 mm, and thosefor (I–L) 8 mm.

Minocycline Inhibits Brain Trauma 1395


resemble neurons, but the cell-specific expression of caspase-1and caspase-3 remains unknown (8). Here we demonstrate,using antibodies recognizing the active form of caspase-1 andcaspase-3, that they colocalize with the neuron specific markerNeuN within the traumatized tissue (Figs. 1 and 2, panels Hand L). Some neurons in the traumatic area contain caspase-1and caspase-3 in the nucleus, which is consistent with previ-ous observations by others indicating translocation of bothproteins into the nucleus in the terminal phases of apoptosis(25, 27). Caspase-1 contains a nuclear localization domain,and its translocation to the nucleus during apoptosis has beendescribed in vitro (27). Since caspase-1 and caspase-3 areexpressed in neurons after TBI, and minocycline inhibits theirup-regulation we investigated whether minocycline coulddemonstrate beneficial effects in experimental TBI in mice.

Minocycline ameliorates neurological dysfunctionafter TBI

Mice undergoing TBI had the expected motor deficits, asassessed by Rotarod performance, when compared with micethat underwent craniotomy without TBI. Mice receiving in-traperitoneal injections of minocycline before or after TBI hadsignificantly decreased motor deficits compared with saline-treated mice (Fig. 3). Saline-treated animals that underwentTBI were able to stay on the rotating rod at 25 rpm for 4.2 60.7 minutes 4 days after trauma compared with 9.1 6 1.1 and6.4 6 0.8 minutes for mice receiving minocycline pretreatmentor posttreatment, respectively. All sham-operated mice wereable to remain on the rotating rod at 25 rpm for 10 minutes, atwhich point the test was ended. The improved neurologicalperformance observed in minocycline-treated animals after

TBI was detected as early as 24 hours and was maintainedthroughout the evaluation period.

Minocycline decreases lesion size after TBI

We then evaluated whether improved motor performancein minocycline-treated mice correlated with decreased post-traumatic lesion size. Four days after the injury, lesion sizewas evaluated by Hematoxylin and Eosin staining as previ-ously described (12). Saline-treated mice that underwent TBIhad a lesion volume of 64.7 6 7.5 mm3 compared with 31.4 63.4 mm3 (P , 0.01) and 35.5 6 4.6 mm3 (P , 0.05) for micereceiving minocycline pretreatment or posttreatment, respec-tively (Fig. 4). The degree of TBI-mediated tissue injury cor-relates with the observed neurological dysfunction.

Minocycline decreases mature IL-1b productionafter TBI

Given that caspase-1 plays a critical role in the progressionof secondary injury resulting from traumatic brain injury andthat minocycline attenuates the expression of several genesincluding caspase-1, we sought to determine whether mino-cycline treatment results in a reduction of caspase-1 activity.Caspase-1 activity in brain sections of the ipsilateral hemi-sphere, as measured by the production of mature IL-1b, wasup-regulated in mice after TBI (154.7 6 11.0 pg/g brain tissue)compared with sham-operated mice (22.4 6 1.2 pg/g braintissue) (Fig. 5). These findings confirmed our previous workdemonstrating posttraumatic caspase-1 activation (12). Mino-cycline treatment significantly decreased mature IL-1b pro-duction to 99.5 6 6.4 pg/g brain tissue (P , 0.002). The factthat inhibition of caspase-1 activity in cerebral ischemia, neu-

FIGURE 2. Activation ofcaspase-3 after TBI.Brain sections of sham-operated mice (A–D).Brain sections 24 hoursafter TBI (E–H) at lowmagnification. Brain sec-tions 24 hours after TBI(I–L) at high magnifica-tion. Caspase-1 stainingis evident in the neuro-nal cytoplasm andnucleus. Size bars for(A–D) and (E–H) repre-sent 20 mm, and thosefor (I–L), 8 mm.



rodegenerative diseases, and traumatic brain and spinal cordinjury results in significantly improved neurological functionin vivo and decreased apoptosis at the cellular level under-scores the significance of caspase-1. The ability of minocyclineto inhibit caspase-1 activity likely contributes to its ability toimprove neurological performance and decrease tissue injuryin this model of TBI.

DISCUSSION

Previous work by our laboratory and others has implicatedcaspase-1 and caspase-3 involvement in TBI. Nevertheless theexact cell-type localization of active caspase-1 and caspase-3

was not known. Caspase-3 had been colocalized with TUNEL-positive cells in the hippocampus after TBI in rats but thespecific cell type was only determined morphologically (8).We show for the first time the localization of activatedcaspase-1 and caspse-3 in neuronal cells after brain trauma.This suggests that neurons undergoing apoptosis secondaryto TBI do so through a mechanism involving both caspase-1and caspase-3.

Minocycline, a tetracycline derivative, has been widelyused as an antibiotic to treat acne and some staphylococcalinfections (37). More recently tetracycline and its derivativeshave been shown to have anti-inflammatory properties (2, 7,33, 46, 47). In a placebo-controlled, double-blind study mino-cycline demonstrated some benefit for patients with rheuma-toid arthritis (42). Others have demonstrated that minocyclinedecreases the expression of caspase-1 and iNOS in cerebralischemia in mice (46, 47). In a mouse model of Huntington’sdisease, caspase-3 is down-regulated in addition to caspase-1and iNOS after minocycline treatment (7). The mechanism ofminocycline inhibition of apoptosis and inflammation is atleast partially due to its inhibition of mature IL-1b production.It is clear that caspases are involved in the induction andexecution of programmed cell death in acute and chronicneurological disorders (7, 11–13, 19, 24–26, 32, 34, 45, 49). Theexact mechanism by which caspase-1 mediates apoptosis isnot entirely clear. Expression of a dominant negative mutantof caspase-1 (encoding ICEC285G) under the control of theneuron specific enolase promotor (NSE) in the NSE-M17Zmouse confers tissue protection after acute neurological in-sults such as ischemia, traumatic brain injury, and spinal cordinjury, as well as chronic neurodegenerative diseases such asamyotrophic lateral sclerosis, Huntington’s disease and Par-kinson’s disease (12, 15, 18, 24–26, 32). A similar effect isobserved with pharmacological inhibition of caspase-1 usingselective peptide inhibitors (12, 19). In certain cell lines, IL-1bis capable of mediating apoptosis independent of caspase-1

FIGURE 3. Minocycline improves motor function after TBI.Mice pretreated with minocycline did significantly betterthan saline-treated mice at 1 to 4 days after injury. Posttreat-ment with minocycline improved motor function significantlyat 2 to 4 days after TBI, albeit to a lesser extent than pre-treatment. P < 0.05 for pretreatment group 1 to 4 days, andposttreatment group 2 to 4 days after TBI (n 5 8 for allgroups).

FIGURE 4. Minocycline reduces TBI-mediated tissue injury.Mice receiving minocycline before or after TBI had signifi-cantly decreased lesion size when compared to saline-treatedmice. The reduction in lesion size was more pronouncedwith minocycline pretreatment (n 5 4 for mice receivingminocycline, n 5 8 for saline-treated group).

FIGURE 5. Minocycline treatment reduced TBI-mediatedcaspase-1 activity. TBI-mediated caspase-1 activation as mea-sured by mature IL-1b production was significantly reducedin mice receiving minocycline when compared with saline-treated mice (n 5 4 for all groups).



activity suggesting IL-1b function at least partially accountsfor caspase-1 involvement in apoptosis (3). Indeed, intracere-bral injections of IL-1b into rats can induce inflammation andapoptosis without any neurological insult (22). The inhibitionof caspase-1 by any of these mechanisms or by minocycline iseffective in decreasing the degree of apoptosis and microglialactivation from many neurological insults. Whether these ef-fects occur through caspase-1-mediated cleavage of the effec-tor caspase-3, production of IL-1b, or some other mechanismis not entirely known at this time (10, 39). The role ofcaspase-3 in apoptosis has been better delineated and it hasbeen shown to act as an executioner or terminal caspase (40,49). It mediates apoptosis by cleaving several targets includ-ing caspase-activated DNase (CAD), nuclear lamins, fodrin,gelsolin, and p21-activated kinase (PAK)-2, that are thought toplay essential roles in apoptotic cell death (20). Capsase-3 canbe activated by caspase-1, caspase-8, caspase-9, andcaspase-11 (10, 20, 23, 39).

In this study we demonstrate, for the first time, TBI-mediated neuronal caspase-1 and caspase-3 activation. Wethen demonstrate that mice treated with minocycline beforeor after TBI demonstrate improved performance on Rotarodwhen compared with saline-treated animals 1 to 4 days aftertrauma. This result indicates that minocycline is an effectivepharmacological agent resulting in improved neurologicalfunction after TBI in vivo. We also show that the improvedneurological function correlates with decreased lesion sizeand decreased caspase-1 activity, as measured by matureIL-1b production. Pretreatment with minocycline providedbetter protection but treatment 30 minutes after trauma wasalso significantly better than saline-treated animals. This dataand the clinical proven safety of minocycline provide therationale to test the drug in human trauma patients.

ACKNOWLEDGMENTS

ROSM and VOO contributed equally to this work. Wethank Junying Yuan (Harvard Medical School) for providingthe caspase-1 antibody and Anu Srinivasan for providing theactive-caspase-3 (CM1) antibody (Idun Pharmaceuticals). Thiswork was supported by the National Institutes of Health andby a departmental grant.

Received, January 17, 2001.Accepted, March 1, 2001.Reprint requests: Robert M. Friedlander, M.D., Division of Neurosurgery,Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street,Boston, MA 02115. Email: [email protected]

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COMMENTS

The authors report that after traumatic brain injury in micethere is activation of caspase-1 and caspase-3 in neuronal cells



that may contribute to secondary CNS injury. They utilizeimmunohistochemical staining techniques that show evidencethat antibodies that recognize active forms of caspase-1 andcaspase-3 colocalize in cells that recognize neuronal specificmarker (NeuN) within neurons in the traumatic lesion. Theauthors propose that minocycline is capable of inhibitingcaspase-1 activation and thus prevents apoptosis and second-ary neuronal injury. In addition to its antibacterial properties,minocycline is known to prevent apoptosis in a number ofexperimental paradigms. Animals that received minocyclineeither 12 hours before injury or 30 minutes after injury dem-onstrated reduced activation of caspase-1, a significant de-crease in size of the traumatic brain lesion, and improvedmotor skills upon testing with Rotarod studies. Althoughdramatic results were observed when the agent was given 12hours before injury, the effectiveness of minocycline as aneuroprotective agent would be limited, if there were not asignificant time window after injury in which the drug couldbe given and still provide benefit. Indeed when given 30minutes after injury, there still appeared to be significantimprovement in motor skills based on the Rotarod test com-pared with saline-treated animals. Further work needs to bedone to establish the limits of the drug’s efficacy with dose-response and time-course studies.

The authors propose that minocycline may be clinicallyuseful in reducing the extent of neuronal injury after headtrauma in humans. The use of minocycline as a neuroprotec-tive agent is attractive for several reasons. A neuroprotectiveagent should have several characteristics including a windowof efficacy after trauma, lack of toxic side effects, the ability todeliver the agent in a simple matter such as systemic admin-istration that may be used in the field setting, and reasonableexpense. Minocycline has a history of safety in clinical use.The above report suggests it may have an effect in ameliorat-ing traumatic brain injury. It would be reasonable to considera protocol for testing in human trauma patients.

Ralph G. Dacey, Jr.Keith RichSt. Louis, Missouri

The authors have presented comprehensive work demon-strating that a “nonexperimental” and widely used and prob-ably safe agent, minocycline, ameliorates the effect of exper-imental TBI in mice. The paper contains an “explanatory” endpoint (2). Yes, indeed, minocycline does what it is supposed todo, i.e., it diminishes mature IL-1b in TBI. It also contains“pragmatic” end points: it diminishes lesion volume and im-proves neurological outcome. Moreover, a beginning of time-window response curves (3) is described: pretreatment ismore effective than treatment begun 30 minutes after theinjury, but the latter is still rather effective. Thus, we concurwith the authors that minocycline treatment appears to beattractive to test with human trauma patients, but to do soprematurely might well hurt its chances to be found effective(1).

First, better dose-response and time-window responsecurves need to be established, initially for explanatory andthen for pragmatic end points.

Second, is there an optimum length of treatment, consider-ing that short term treatment interferes with a “destructive”cascade, but that prolonged treatment may interfere withrestorative and repair mechanisms?

Third, the model used in this paper is a contusion model,and the pragmatic outcome measures, i.e., lesion volume andmotor function, are specific to this model; thus, will it alsowork in a “diffuse” model of brain injury affecting cognitiveoutcome measures?

And finally, does it also work in other species?There have been many reasons why in the past new models

of treatment have not been sufficiently tested in animals be-fore being applied to humans, not the least of which is thatpharmaceutical companies often feel compelled to test newdrugs as rapidly as possible due to the limited patent protec-tion period. This does not apply to minocycline, so let us givethis drug all the chances it deserves by doing thorough pre-clinical testing before trying it in humans.

J. Paul MuizelaarSacramento, California

1. Bullock MR, Lyeth BG, Muizelaar JP: Current status ofneuroprotection trials for traumatic brain injury: Lessons fromanimal models and clinical studies. Neurosurgery 45:207–217,1999.

2. Schwartz D, Lellouch J: Explanatory and pragmatic attitudes ther-apeutically trials. J Chronic Dis 20:637–648, 1967.

3. Verweij BH, Muizelaar JP, Vinas FC, Peterson PL, Xiong Y, Lee CP:Improvement in mitochondrial dysfunction as a new surrogateefficiency measure for preclinical trials: Dose-response and time-window profiles for administration of the calcium channel blockerZinconotide in experimental brain injury. J Neurosurg 93:829–834,2000.

The authors of this paper tested directly several hypothe-ses. First, they show that caspase-1 and caspase-3 are in-creased by trauma and that these factors are localized inneurons. Second, they show that both pretreatment and post-treatment of the injured animals with minocycline decreaseslesion size and improves a measure of neurological function.Further minocycline treatment decreases tissue levels of IL-1b. Together, this is potent evidence confirming the role ofcaspase activation and apoptosis in the response to braininjury. The authors conclude that the use of minocycline in aclinical trial would be appropriate.

This is an excellent paper that solidifies the role of caspaseactivation and apoptosis in participating in the anatomic andfunctional effects of brain injury. An intriguing aspect of thiswork is that minocycline is already in daily use, makingdevelopment of a clinical trial more straightforward thanusual. Of course, when the usual range of neurosurgical pro-cedures is considered, it is apparent that brain trauma is acommon thread in determining outcome and that any maneu-ver that can reduce the effects of that trauma, whether byretraction or ischemia, will be a boon for our patients.

Charles J. Hodge, Jr.Syracuse, New York



The authors compared intraperitoneal minocycline versussaline in a rodent model of traumatic brain injury (TBI). Theantibiotic minocycline has been found to down-regulatecaspase-1, inducible nitric oxide synthase and to have anti-inflammatory properties. In this study, caspase-1 andcaspase-3 colocalized to neurons in the affected area after TBI.Minocycline-treated animals were found to have decreasedmotor deficit, decreased lesion size and decreased IL-1b pro-duction. The authors believe that these results and clinicalexperience with minocycline provide rationale for humantrials in trauma patients.

This is a well-designed and reported study. We agree that ifthese results are verified, minocycline, a drug that has beenemployed for important but less debilitating conditions,should be considered for human trials. Although this studymakes a compelling argument that minocycline amelioratesbrain injury through caspase-1 inhibition, other properties ofthis drug noted by the authors or unknown effects may alsobe important.

Christopher L. TaylorWarren R. SelmanCleveland, Ohio

This is an interesting study in mice that presents somepreliminary evidence that minocycline may be an effectiveneuroprotectant after traumatic brain injury. The authors pro-pose that minocycline reduces neuronal loss because it de-creases expression of caspase-1 and caspase-3, thereby block-ing apoptotic pathways. Based on the findings of this study,and the proven safety of minocycline in humans, the authorspropose that this agent should now be tested in human headinjury patients.

Although use of minocycline may ultimately prove to beeffective for human head injury, embarking on clinical trials atthis time may turn out to be somewhat premature for at least

two reasons. First, the relatively short 4-day postinjury sur-vival period before histological assessment, leaves open thedistinct possibility that minocycline may have only delayedeventual neuronal degeneration. Recent studies, e.g., byPierce et al. (1), have shown that trauma-induced neuronaldegeneration is an ongoing process, which may take up to ayear to fully manifest. Although it is unreasonable to expectall studies to wait that long, a longer acute survival period inthis study, such as 3 to 4 weeks, would have provided aclearer picture as to the ultimate effect of this agent. Second,the diminished effectiveness of the agent in improving neu-ronal function and lesion size when given only 30 minutespostinjury, compared with preinjury administration, raisesthe question of whether its effectiveness will be lost whengiven in a more clinically relevant time such as 2, 4, or even 8hours postinjury. Over the last decade, there have been nu-merous neuroprotectants when given preinjury or immedi-ately postinjury that have shown great promise under idealcircumstances. It would be optimal to see more convincingdata that the therapeutic time window is sufficiently wide inthis animal model before human testing.

Overall this is a well-done and important preliminarystudy. Hopefully the authors will expand their investigationsto provide more convincing data that this agent is ready forhuman clinical trials.

Daniel F. KellyStefan M. LeeLos Angeles, California

1. Pierce JE, Smith DH, Trojanowski JQ, McIntosh TK: Enduringcognitive, neurobehavioral and histopathological changes persistup to one year following severe experimental brain injury in rats.Neuroscience 7:359–369, 1988.

From, J. (Jean) Cruveilhier’s Anatomie pathologique du corps humain, ou Descriptions, avec figures lithographiées et coloriées, desdiverses altérations morbides dont le corps humain est susceptible. Paris, Baillière, vol. 2, 1829–1842. (Courtesy, Rare Book Room,Norris Medical Library, Keck School of Medicine, University of Southern California, Los Angeles, California.)


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