Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia*

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-d-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

Stroke is one of the most common life-threatening neurological diseases. Despite significant advances in the understanding of the molecular events following cerebral ischemia, there are still no potent neuroprotective therapeutics against stroke-induced brain damage (1)(2)(3). The ischemia-induced excessive release of neurotransmitter glutamate causes excitotoxicity, which is believed to be the major cause of toxicity to neurons (1,4,5). Glutamate overactivates NMDA 2 receptors, causing increased intracellular Ca 2ϩ influx leading to the accumulation of toxic levels of intracellular calcium ions (4,5). Elevation in intracellular Ca 2ϩ concentrations activates Ca 2ϩ -dependent proteases, such as calpains, which break down critical structural proteins causing neuronal death (3, 6 -9).
Chemical compounds directly blocking glutamate toxicity to neurons may have the potential to be developed as therapeutics to stroke. But NMDA receptor blockers, such as MK-801, have failed in human stroke clinical trials due to the severe side effects possibly resulting from interference with the normal physiological functions of the NMDA receptor, despite the fact that compounds like MK-801 are very effective in preventing glutamate-mediated neuronal death in cell culture models (2). Tetracyclines are antibiotic agents with a broad spectrum of anti-microbial activities and anti-inflammation properties (21). Recent studies demonstrated that minocycline, a tetracycline derivative, has potent neuroprotective properties in animal models of various brain diseases, such as global and focal cerebral ischemia (21)(22)(23), spinal cord injury (24), retinal cell death (25), Parkinson disease (26), Huntington disease (27), multiple sclerosis (28), and amyotrophic lateral sclerosis (29). The potential mechanisms of minocycline-mediated neuroprotection are through suppression of microglial activation and inhibition of the release of apoptotic factors such as cytochrome c and attenuation of intracellular caspase activities (21,27,29). However, it is still not clear whether minocycline interferes with NMDA receptor function. Tetracyclines, in general, have been used safely as an antibiotic agent for many years in the clinic. The properties of clinical tolerance and easy penetration into the brain make some of the tetracycline derivatives potential therapeutic reagents for neuroprotection in stroke (30 -33).
In the present study, we tested the protective effects of two tetracyclines against glutamate-mediated excitotoxicity and cerebral ischemiainduced brain damage. The potential molecular mechanisms of such neuroprotection were also investigated. CTC and DMC were found to be strongly neuroprotective, not through inhibition of the NMDA receptor but rather through suppression of a Ca 2ϩ rise and inhibition of calpain activities.

Primary Cultures of Cerebellar Granule Neurons (CGNs)
Primary cultures of mouse (C57/B6) CGNs were prepared from 6-to 9-day-old postnatal mice as described previously (34,35). Briefly, cerebella were explanted and cleaned free of meninges. Mechanical and enzymatic dissociation in a 0.025% w/v trypsin solution for 25 min followed. A trypsin inhibitor was then added to block the enzyme, and 0.05% w/v DNase was added to break DNAs from dead cells. A series of trituration and mild centrifugation steps were included to disperse the neurons prior to resuspension in medium and to remove undissociated debris prior to plating in Eagle's minimum essential medium containing 0.8 mM glutamine, 27 mM glucose, 0.01% gentamycin, 9% fetal bovine serum and supplemented with K ϩ to a final concentration of 23 mM. Cells were plated onto 24-well dishes containing poly-L-lysine-coated coverslips at a density of 6 ϫ 10 5 per well. After ϳ18 h, cytosine ␣-Darabinofuranoside (AraC) was added to a final concentration of 5 M, to prevent glial cell proliferation. 100-mm dish cultures were seeded with 21 ϫ 10 6 cells in 10 ml of culture medium.

Neuronal Viability Assays
Tetracyclines were added to 8-day in vitro cultured CGNs at 37°C for 15-20 min prior to treatment with 50 M glutamate or NMDA. The plates were then incubated for 6 h at 37°C. Untreated controls were also included. At the end of the treatment period, neuronal viability was measured using the CFDA assay as described previously (34,35). The CFDA stock solution was diluted using Earle's balanced salts (Sigma) to a final concentration of 5 g/ml. Cultures were incubated with 500 l of the CFDA solution at 37°C for 30 min. The intensities of fluorescence was quantified using a Cytofluor TM 2350 Fluorescence Measurement System (Millipore) at ex ϭ 480 nm and em ϭ 530 nm. Cellular viability was normalized against the fluorescent reading from the control cells. Neurons were also fixed in 4% formaldehyde and mounted in Dako® fluorescent mounting medium containing 5 g/l Hoechst 33258 to detect nuclei under a fluorescent microscope. Duplicate assessment of each treatment was made on each plate in at least three separate experiments per treatment.

Whole-cell Recording
Whole-cell recording using electrophysiological methods has been described previously (36). Briefly, CGNs, cultured on 35-mm culture dishes, were perfused continuously at 1 ml/min at 22°C using a solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 10 mM HEPES, 3 mM glucose, pH 7.4. The perfusion solution also contained 1 M tetrodotoxin, 30 M glycine, and 1 M strychnine. Patch pipettes (2-4 M⍀ resistance) were constructed from 1.5 mm outer diameter/1.0 mm inner diameter Pyrex 7740 glass (Corning, Big Flats, MN). A modified DAD-12 perfusion system (ALA Scientific Instruments, Westbury, NY) was used to rapidly apply NMDA (2-s duration) followed by co-application of NMDA and the test compound (5-s duration). The pipette solution contained 140 mM CsCl, 1.1 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP at pH 7.2. Whole-cell currents were acquired using an Axopatch 1-D amplifier equipped with a CV-4 head stage with a 1 G⍀ feedback resistor (Axon Instruments, Foster City, CA). Voltage command and current acquisition were accomplished using a Digidata 1200 interface and pClamp 6.0 software (Axon Inst). Neurons were held at a membrane potential of Ϫ60 mV. The fractional block of NMDAevoked currents was calculated according to the formula: B ϭ I Ϫ I B /I, where I is the steady-state current evoked by NMDA, and I B is the current evoked by NMDA in the presence of the test compound at the end of the co-application.

Intracellular Ca 2؉ Measurement
Fluo-4 Measurement-Intracellular calcium concentration was measured as described previously (37). Briefly, culture medium in the 24-well plate was replaced with a calcium sensitive dye Fluo-4 (4.5 M) in a balanced salt solution. After 30-min incubation, the dye was removed and cells were incubated with the original medium with or without the compound at 37°C for 15 min. Fluorescent intensities were quantified using a Cytofluor TM 2350 Fluorescence Measurement System (Millipore) at ex ϭ 485 nm and em ϭ 530 nm. NMDA (50 M) was then added to the wells and changes in fluorescence were recorded after 5, 10, 20, 30, and 40 min. The fold increase in Ca 2ϩ was calculated by subtracting the initial reading from each reading divided by the initial reading.
Ratiometric Measurement of [Ca 2ϩ ] Using Fura-2-To quantitatively determine the effect of CTC or DMC on glutamate-induced changes in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) level, ratiometric measurement of [Ca 2ϩ ] i was performed using fura-2 AM. Briefly, mouse CGNs at 7 days in vitro on glass coverslips were loaded with 5 M fura-2-AM (Molecular Probes) plus 0.02% pluronic (Molecular Probes) for 30 min at 37°C. After rinsing with PSS Mg 2ϩ -free buffer containing 2 mM HEPES (pH 7.2), 140 mM NaCl, 5 mM KCl, 2.3 mM CaCl 2 , and 10 mM glucose, and stabilized in the same buffer for 5 min, fura-2 intensities were measured using a Northern Eclipse Digital Ratio Image System (Empix, Mississauga, Ontario, Canada) with Axiovert 200 camera and light source (Zeiss, Thornwood, NY). Fura-2 fluorescence was measured at 510 nm emission with 340/380 nm dual excitation selected by a DG-5 system (Sutter Instrument Co., Novato, CA). Changes in [Ca 2ϩ ] i concentration was measured by converting the 340/380 ratio of fura-2 florescence (after correction for background) to approximate [Ca 2ϩ ] i using the method as described by Grynkiewicz et al. (38) and Young et al. (39). The 340-to-380 nm fluorescence ratio (R340/380) for 20 cells in one field of each coverslip was averaged. The minimal and maximal fluorescence ratios (R min and R max ) were obtained from a sample set of CGNs using 5 M ionomycin plus 6 mM EGTA and by 10 mM CaCl 2 , respectively. The K d for fura-2 was assumed to be 224 nM as described by Young et al. (39). The basal level of [Ca 2ϩ ] i was recorded for 10 s, followed by the application of the compound dissolved in PSS buffer, which also contains 10 M glycine for another 40 s, and finally glutamate (50 M) was applied, and the recording was continued for another 200 s. All measurements were repeated for at least three times. The data were analyzed using Microsoft Excel.

Animal Ischemia Surgery
All procedures using animals were approved by the Institute of Behavioral Science Animal Care Committee following the guidelines established by the Canadian Council on Animal Care. C57B/6 mice (20 -23 g) were obtained from Charles River and bred locally. Under temporary isofluorane anesthesia, mice were subjected to MCAO using an intraluminal filament as previously described (40,41). After 1 h of MCAO, the filament was withdrawn, blood flow restored to normal by laser Doppler flowmetry, and wounds sutured. Mice were injected with CTC or DMC intraperitoneal at 90 mg/kg body weight 4 h before ischemia, followed by injection twice per day. The control groups included no treatment or vehicle in which animals were injected with the same volume of saline. Brains were removed after 24-h reperfusion, and the brain infarction was measured as described as follows.

Infarct Size Measurement
Infarct size was measured by a colormetric staining method using 2,3,5-triphenyltetrazolium chloride (TTC) as described previously (40,41). Briefly, brains were dissected out and cut into four 2-mm-thick coronal slices, which were stained with 5 ml of 2% TTC for 90 min at 37°C. Afterward, the tissue was rinsed with saline and subsequently exposed to a mixture of ethanol/dimethyl sulfoxide (1:1), which was to solubilize the formazan product. After 24-h incubation in the dark, the red solvent extracts were diluted 1:20 with fresh ethanol/Me 2 SO solvent in three tubes and placed in cuvettes. Absorbance was measured at 485 nm in a spectrophotometer and the values were averaged. Percentage loss in brain TTC staining in the ischemic side of the brain was compared with the contralateral side of the brain of the same animal using the following equation: % loss ϭ (1 Ϫ (absorbance of ischemic hemisphere/absorbance of contralateral hemisphere) ϫ 100).

Neurological Scores
An expanded six-point scale was modified based on previous reports (40 -42) and used for the present investigation. Behavioral assessments were made at 0 and 24 h after reperfusion by an individual blinded to the treatment of the mice. The neurological deficits were scored as follows: 0, normal; 1, mild turning behavior with or without inconsistent curling when picked up by tail, Ͻ50% attempts to curl to the contralateral side; 2, mild consistent curling, Ͼ50% attempts to curl to contralateral side; 3, strong and immediate consistent curling, mouse holds curled position for more than 1-2 s, the nose of the mouse almost reaches tail; 4, severe curling progressing into barreling, loss of walking or righting reflex; 5, comatose or moribund. At least eight mice per group were evaluated for each compound and scores were averaged for statistical analysis.

In Vitro Measurement of Calpain Activity
Calpain activity was measured using a calpain activity assay kit (Calbiochem, Mississauga, Ontario, Canada) following the manufacture's instructions. The assay is based on fluorometric detection of cleavage of calpain substrate Ac-Leu-Leu-Tyr-AFC using a Cytofluor TM 2350 Fluorescence Measurement System (Millipore). The cleavage resulted in the release of AFC that can be measured in a fluorometer. Briefly, constitutive calpain I or II (0.1 unit/ml) (purchased from Calbiochem) was activated by 500 M Ca 2ϩ and was mixed with CTC (150 M), DMC (150 M), minocycline (150 M), or calpain inhibitors ALLN or calpastatin (10 M each) and 5 l of calpain substrate to a final volume of 100 l. The mixture was incubated at 37°C for 1 h in the dark. The cleavage of the substrate resulted in the release of AFC that can be detected by the Cytofluor at ex ϭ 400 nm and em ϭ 505 nm.

Western Blotting
Protein at 10 g was electrophoresed in a 7% SDS mini gel and then electroblotted onto a nitrocellulose membrane in transfer buffer (39 mM glycine, 48 mM Tris base, and 20% methanol) as described previously (34). The membrane was then probed with a polyclonal antibody selective to calpain cleaved fragment of brain spectrin at 4°C overnight. After washing with 0.01 M phosphate-buffered saline, horseradish peroxidase-conjugated secondary antibody was applied to the membrane for 1 h at room temperature. Enhanced chemiluminescence detection of the target protein was performed using a LumiGlo substrate kit (KP Laboratories, Gaithersburg, MD) and x-ray film.

Data Analysis
Data were analyzed using Microsoft Excel and Prism. Statistical significance was determined by Student's t test, and the significant group was determined using further post hoc Tuckey's test. p Ͻ 0.05 was considered statistically significant.

CTC and DMC Protect Cultured CGNs against Glutamate
Toxicity-The neuroprotective effects of CTC, DMC, and minocycline against glutamate-mediated excitotoxicity in cultured mouse primary CGNs were examined and quantified using the CFDA assay. All three compounds showed potent neuroprotection against glutamate-mediated toxicity to CGNs in a dose-and time-dependent manner (Fig. 1, A-C). More than 85% of the CGNs were protected by these two compounds at doses ranging between 80 and 150 M. This protection lasted up to 8 h following glutamate treatment when more than 50% of the control CGNs was killed by glutamate. The appearance of dead neurons was visualized by Hoechst staining of the nuclei (Fig. 1C). CTC and DMC were not toxic to CGNs at the ranges of doses tested (data not shown).
CTC and DMC Reduce MCAO-induced Brain Damage-Since minocycline has been shown to provide neuroprotection against cerebral ischemia, we examined the neuroprotective effect of CTC and DMC in a mouse model of focal ischemia with 1 h MCAO followed by 24-h reperfusion. Each compound was administered by intraperitoneal injection 4 h prior to MCAO at 90 mg/kg and followed by two more injections (8 and 16 h following reperfusion) at 45 mg/kg. Animals were then killed to remove the brain for analysis as described under "Experimental Procedures." Both compounds significantly reduced the infarct size in the cerebral cortex by almost 50% in comparison with the non-treated ischemic control and vehicle-treated brain (p Ͻ 0.05, Fig. 2, A and B). Coronal sections of the brain slices numbered as 1-4 were shown in Fig.  2, B-D. Most of the infarctions occurred in the first two brain slices in the cerebral cortex and striatum as indicated by the arrows in Fig. 2B. The infarction was significantly reduced in the same areas in brains treated with CTC (Fig. 2C) or DMC (Fig. 2D).
The protective effects of these compounds were also confirmed by the improvement of the neurological behavior of the compound-treated ischemic mouse. Using the six point valuation system as described in the "Experimental Procedures" section, the scores of the neurological behavior of ischemic animals were compared with those of vehicletreated or ischemic animals 0.5 h after surgery and after 24-h reperfusion. As shown in Fig. 2E, mice treated with the two compounds showed significant improvement after 24 h of reperfusion (p Ͻ 0.05) compared with the vehicle-treated or ischemic animals, demonstrating that CTC and DMC reduced MCAO-induced neurological deficits.

CTC and DMC Weakly Antagonize NMDA Receptor Activity and
Suppress the Rise in Intracellular Ca 2ϩ -To understand the mechanisms of neuroprotection conferred by CTC and DMC, we examined whether these two compounds blocked calcium entry through the NMDA receptor, which has been implicated in mediating glutamateinduced exitotoxicity. Both compounds at 150 M showed weak, but rapid, antagonism to 50 M NMDA-induced currents (Fig. 3A). A 5 s co-application of NMDA plus 150 M CTC resulted in a 14 Ϯ 1% (n ϭ 5) reduction in NMDA-induced current. A 5 s co-application of NMDA plus 150 M DMC produced a 16 Ϯ 2% (n ϭ 5) reduction in NMDAinduced currents.
Since NMDA activation induces intracellular Ca 2ϩ influx, we next tested whether these two compounds affect glutamate and NMDAinduced intracellular Ca 2ϩ levels. As shown in Fig. 3B, NMDA receptormediated intracellular calcium influx increased immediately after the addition of glutamate. The addition of the two compounds partial blocked [Ca 2ϩ ] i influx, but the [Ca 2ϩ ] i level eventually increased to the same level as that of NMDA-treated CGNs after 40 min (Fig. 3C). Minocycline also exhibited a similar level of blockade of Ca 2ϩ influx as compared with those from CTC and DMC at the 10-and 20-min time points (Fig. 3C, p Ͻ 0.05 compared with NMDA-mediated Ca 2ϩ rise). On the other hand, MK-801, an antagonist to NMDA receptor, completely blocked glutamate and NMDA-induced Ca 2ϩ influx (Fig. 3, B and C). Taken together, these data demonstrated that CTC and DMC are weak and transient blockers of the NMDA receptor currents and only partially block [Ca 2ϩ ] i influx during the early stages of glutamate/NMDA treatment. However, such a transient reduction in NMDA receptor current and [Ca 2ϩ ] i influx may not be sufficient to account for the more than 85% neuroprotection conferred by these two compounds, suggesting that these compounds may inhibit intracellular targets.
CTC and DMC Protect CGNs through Inhibition of Intracellular Calpain Activities-Calcium-activated intracellular proteases such as calpain are an important mediator of neuronal death in response to glutamate toxicity and cerebral ischemia (1). Although caspase activity may also play a role in the apoptotic component of ischemia-induced neuronal death, our previous work showed that caspase is not active in glutamate-induced neuronal death (1). To understand how CTC and DMC protect neurons in vitro and in vivo, we hypothesized that these two compounds could modulate the activities of Ca 2ϩ -activated calpains.
To do this, in vitro experiments were first performed using purified exogenous calpains. CTC and DMC significantly inhibited the activities of active calpain I (Fig. 4A, p Ͻ 0.001) and calpain II (Fig. 4B, p Ͻ 0.001).   1-4 in B-D indicate the first to the last slice of the MCAO brain and arrows indicate ischemic infarction (white-colored region on the brain slice). E, the respective scores of neurological behavior of each mouse were plotted and presented. * indicates statistical significance (p Ͻ 0.05) by Student's t test. C is a graph indicating that CTC and DMC partially block [Ca 2ϩ ] i influx as measured by Fluo-4 assay. CGNs were treated with or without the compound indicated in the graph and followed by NMDA application. Intracellular calcium concentrations were measured using Fluo-4 as described under "Experimental Procedures." The fold of increase was calculated against non-treated CGNs. At least three independent repeats were performed, and data presented are mean Ϯ S.E.; ** indicates p Ͻ 0.01, and * indicates p Ͻ 0.05 when compared with NMDA.
Specific inhibitors to calpains (ALLN and calpastatin), which inhibited calpain activity and also prevented neuronal death, were used as positive controls for the assay. Interestingly, minocycline, a potent neuroprotectant, did not inhibit the activities of calpains (Fig. 4, A and B).
Next, we examined whether these two compounds could inhibit glutamate-induced activation of calpains in CGNs. Calpain activity was monitored by the presence and the level of the SBP on Western blot. As shown in Fig. 4, C and D, after 20-min treatment with 50 M glutamate, the level of SBP increased significantly (p Ͻ 0.001, Fig. 4D), and the level of SBP reached a peak after 2.5 h (Fig. 4D). Calpain inhibitors, ALLN, CTC, and DMC, were applied to cultured CGNs 30 min prior to glutamate treatment. Both the calpain inhibitor, and the two compounds significantly reduced the level of SBP caused by glutamate treatment in comparison with glutamate only treated sample at 2.5 h (Fig. 4D, p Ͻ 0.05).
Furthermore, the two compounds CTC and DMC also inhibited calpain activities caused by MCAO in mice brain as shown by the reduced level of SBP on Western blot (Fig. 4, E and F). The SBP level increased sharply in the ischemic brain of vehicle-treated mouse, but the level of SBP was significantly reduced in CTC-and DMC-treated brains (Fig. 4, E and F, p Ͻ 0.05). Taken together, CTC and DMC inhibit calpains activation in response to excitotoxicity and cerebral ischemia.

DISCUSSION
In the present study, we report the findings that CTC and DMC are neuroprotective against glutamate toxicity in cultured mouse CGNs in vitro and focal cerebral ischemia in vivo through inhibition of calpains, a mechanism different from that of minocycline. To the best of our knowledge, the present study is the first demonstration that CTC and DMC conferred neuroprotection through inhibition of calpain activities.
The molecular targets of CTC and DMC appear to be downstream of the NMDA receptors. CTC and DMC only weakly inhibited NMDAinduced intracellular calcium influx. The 14 -16% inhibition of NMDA receptors by the two compounds could contribute to the subsequent relatively slow increase in intracellular Ca 2ϩ levels seen in the two compound-treated samples; however, it is highly unlikely that this Ca 2ϩ entry could account for the potent neuroprotection conferred by CTC and DMC to glutamate-treated CGNs and suggests that these two com- Purified exogenous active calpains I and II at 0.1 unit/ml each were mixed with calpain-specific inhibitors ALLN or calpastatin at 10 M or the compound as indicated at 150 M concentration. After 30-min incubation with the calpain substrate, the release of fluorescent AFC was recorded using a Cytofluor. The fluorescent unit/g of protein/h was calculated from at least three independent experiments and plotted in A (calpain I) and B (calpain II) (mean Ϯ S.E.). ** indicates statistical significance (p Ͻ 0.01) by Student's t test. C-F show Western blotting and its quantification of SBP produced by the activation of calpain. Glutamate-treated CGNs in the presence or absence of calpain inhibitor or compounds were collected after the time indicated in C. The protein extract was subjected to Western blotting with a primary antibody against SBP. GAPDH was used as protein loading control. The production of SBP was normalized against GAPDH, and the mean Ϯ S.E. was presented in D. Similarly, ischemic brains were collected for Western blotting to detect the production of SBP and the level of it was normalized against GAPDH (E and F). The mean Ϯ S.E. was presented in F. NB, normal brain; C, contralateral side; I, ischemic side of the brain. ** indicates statistical significance (p Ͻ 0.01) by Student's t test.
pounds target downstream intracellular death signal transduction pathways.
Interestingly, the molecular targets of CTC and DMC appeared to be different from those of minocycline in that CTC and DMC inhibit calpain I and II, whereas minocycline does not. Previous studies have demonstrated that minocycline provides in vivo neuroprotection by suppressing microglial activation (21,22). Reports also showed that minocycline directly target intracellular death pathways to protect neurons through blocking cytochrome c release and the subsequent activation of caspase (23,29). The present in vitro studies using cultured CGNs showed that neuroprotection conferred by CTC and DMC came from direct inhibition of calpain activities but not from inhibition of microglial activation, since microglial activation played no role in such acute glutamate-induced neuronal death system. In addition, caspase did not become activated in this system (1). Indeed, in vitro and in vivo experiments, as shown in Fig. 4, clearly demonstrated that CTC and DMC were potent inhibitors of calpains activated in response to both glutamate treatment and MCAO in mice brains. Calpains are major upstream proteases that are activated following ischemic injury to the brain and are responsible for the rapid and sustained induction of spectrin breakdown in the infarct zone (9,20,43). The present study, using several techniques including in vitro calpain activity assay and SBP quantification by Western blotting, demonstrated that calpain activity increased rapidly following treatment with glutamate in cultured CGNs or MCAO and that this induction can be ameliorated by calpain-specific inhibitors, CTC and DMC. This early induction in calpain following cerebral ischemia is consistent with previous reports that calpain-induced SBP could be detected as early as 1 min in the dendrites of pyramidal cells in the CA2/CA3 border zone and 5-10 min in CA1, cortical, and thalamic regions of the ischemic rats (9). In addition to stroke, the activities of calpain are also implicated in a wide range of pathological conditions such as traumatic brain injury, Alzheimer disease, and type 2 diabetes mellitus (44). As a result, several calpain inhibitors have been developed to target these diseases, for example, natural product-based inhibitors and synthetic inhibitors (45). However, none of them have structural similarities to tetracyclines. Thus tetracycline may inhibit calpain activation/activity in a way distinct from those described in the literature and which requires further investigation.
A compound with the potential to become a lead for development as therapeutics to stroke has to meet several requirements, such as 1) that the compound does not interfere with normal physiological functions of glutamate receptors, 2) that the compound targets intracellular death pathways to provide direct protection to neurons, and 3) that the compound should penetrate into the brain freely and have low toxicity. CTC and DMC used in the present study appear to meet all of the above requirements. CTC and DMC have been used clinically as antibiotics for many years. In addition, tetracyclines also have other beneficial activities and properties such as anti-oxidation and anti-glycation (33,46,47). These advantages of tetracyclines make them promising leads for drug development as stroke therapeutics.
However, further modification of CTC and DMC is required. Since these compounds are known potent antibiotics, it is desirable to use them at low doses and for a short period of time. The doses that we used in the current study were 90 mg/kg prior to MCAO and followed by 45 mg/kg with no visible toxicity present, but a very recent report suggests that low dose minocycline (at 3 mg/kg prior to cerebral ischemia and 10 mg/kg afterward intravenously) indeed conferred neuroprotection in MCAO rats (48). Current work is under way to examine the low dose effect of CTC and DMC. In addition, novel derivatives of CTC and DMC, which do not possess anti-microbial activities, are under development.
In summary, a battery of biochemical experiments performed in the present study demonstrated that CTC and DMC, the two clinically used antibiotics, provide neuroprotection not through blocking NMDA receptors but rather by inhibition of calpain activity. Future modification of these two compounds may lead to drugs capable of neuroprotection following cerebral ischemia.