Prevention of hypoglycemia-induced neuronal death by minocycline
© Won et al.; licensee BioMed Central Ltd. 2012
Received: 6 June 2012
Accepted: 14 September 2012
Published: 22 September 2012
Diabetic patients who attempt strict management of blood glucose levels frequently experience hypoglycemia. Severe and prolonged hypoglycemia causes neuronal death and cognitive impairment. There is no effective tool for prevention of these unwanted clinical sequelae. Minocycline, a second-generation tetracycline derivative, has been recognized as an anti-inflammatory and neuroprotective agent in several animal models such as stroke and traumatic brain injury. In the present study, we tested whether minocycline also has protective effects on hypoglycemia-induced neuronal death and cognitive impairment. To test our hypothesis we used an animal model of insulin-induced acute hypoglycemia. Minocycline was injected intraperitoneally at 6 hours after hypoglycemia/glucose reperfusion and injected once per day for the following 1 week. Histological evaluation for neuronal death and microglial activation was performed from 1 day to 1 week after hypoglycemia. Cognitive evaluation was conducted 6 weeks after hypoglycemia. Microglial activation began to be evident in the hippocampal area at 1 day after hypoglycemia and persisted for 1 week. Minocycline injection significantly reduced hypoglycemia-induced microglial activation and myeloperoxidase (MPO) immunoreactivity. Neuronal death was significantly reduced by minocycline treatment when evaluated at 1 week after hypoglycemia. Hypoglycemia-induced cognitive impairment is also significantly prevented by the same minocycline regimen when subjects were evaluated at 6 weeks after hypoglycemia. Therefore, these results suggest that delayed treatment (6 hours post-insult) with minocycline protects against microglial activation, neuronal death and cognitive impairment caused by severe hypoglycemia. The present study suggests that minocycline has therapeutic potential to prevent hypoglycemia-induced brain injury in diabetic patients.
Hypoglycemia occurs in type 1 and type 2 diabetic patients who attempt strict management of their blood glucose levels with insulin or other glucose-lowering drugs [1–3]. Hypoglycemia causes recurrent morbidity in patients, and sometimes results in mortality [4, 5]. Frequent low blood glucose levels in type 1 diabetic patients can lead to the development of hypoglycemia unawareness, which desensitizes patients to symptoms of low blood glucose . Hypoglycemia unawareness may lead to prolonged nocturnal hypoglycemia, causing convulsions and coma, and resulting in sudden death . Severe hypoglycemia, most commonly encountered in diabetic patients who unintentionally self-administer insulin at supratherapeutic doses, can cause potential complications such as irritability, impaired concentration, focal neurological deficits, confusion, drowsiness, coma, seizure, and neuronal death . Under the most severe conditions, hypoglycemic neuronal death occurs in CA1, subiculum and dentate gyrus of the hippocampus, neurons in cortical layers II and III of the cerebral cortex, and the dorsolateral striatum. Hippocampal neurons are particularly important for learning and memory, and impairment of cognitive abilities is the most common sequelae of hypoglycemic coma . We and other groups have reported that hypoglycemia-induced neuronal death is not a simple result of energy failure resulting from low glucose, but the result of activating cell death-related signaling pathways such as glutamate receptor activation, reactive oxygen species (ROS) production and extracellular zinc release [10–12].
Microglia are the major innate immune cells in the brain. Once microglia are activated by neurological damage or systemic inflammatory events, activated microglia release neurotoxic substances such as nitric oxide, ROS, cytokines, and chemokines, and undergo morphological changes from a ramified to an amoeboid shape [13, 14]. As well, the recruitment of various types of peripherally derived inflammatory cells such as neutrophils, T cells, and macrophages can affect brain inflammation following neurological damage . Inhibition of inflammation increases the rates of neuronal survival and improves neurological deficits in several animal models of neurological injury [16, 17]. Although the exact circumstances under which microglial activation is harmful or beneficial are still controversial , some evidence indicates that early phase inflammation caused by acute brain injury can contribute to neuronal death. In a previous study, we showed that hypoglycemic injury-induced microglial activation is affected by body temperature . However, it is unknown whether microglial activation is a major contributing factor in hypoglycemia-induced neuronal death.
Minocycline is a second-generation semi-synthetic tetracycline derivative that possesses improved tissue penetration into the cerebrospinal fluid and the central nervous system, compared to earlier forms. Beside the anti-microbial effect, minocycline has anti-inflammatory and anti-apoptotic effects. It has been shown that minocycline has neuroprotective effects in animal models of neurodegenerative disease including Parkinson’s , Huntington’s  and Alzheimer’s disease , as well as several animal models of neurological disease such as global ischemia , focal cerebral ischemia  and spinal cord injury . These effects are thought to arise through the inhibition of microglial activation, inducible nitric oxide synthase, COX-2 expression or modulation of cytokine expression. In the present study, we tested whether delayed treatment with minocycline could also reduce microglial activation, neuronal death and cognitive impairment induced by severe hypoglycemia.
Materials and methods
Animal surgery and insulin-induced hypoglycemia
Arterial blood glucose change before, during and after hypoglycemia (mM)
After glucose reperfusion
Vehicle group (n = 6)
4.02 ± 0.13
0.40 ± 0.03
7.93 ± 0.16
Minocycline group (n = 6)
4.08 ± 0.12
0.38 ± 0.01
7.90 ± 0.23
Arterial blood pressure change before, during and after hypoglycemia (mmHg)
After glucose reperfusion
Vehicle group (n = 6)
103.34 ± 1.88
185.07 ± 3.29
107.95 ± 3.85
Minocycline group (n = 6)
101.91 ± 1.79
184.29 ± 3.13
106.44 ± 4.27
After hypoglycemic surgery or sham operation, rats in each group were randomly assigned to two subgroups. Minocycline (Sigma, St Louis, MO, USA) dissolved in 0.1 M phosphate buffer, pH 7.4 (50 mg/kg) or vehicle was administered intraperitoneally once daily beginning 6 hours after glucose reperfusion for a duration of 1 week (total of seven times).
At the indicated time points, rats were anesthetized and transcardially perfused with 200 ml of 0.9% saline followed by 200 ml of 4% formaldehyde (FA). The harvested brains were post-fixed for 24 hours and immersed in 20% sucrose. Free-floating coronal sections (40 μm thickness) were incubated with blocking buffer (10% goat serum and 0.1% Triton X-100 in 0.1 M PB) for 30 minutes at room temperature. The sections were then incubated with mouse anti-rat CD11b (1 μg/ml; Serotec, Raleigh, NC, USA), mouse anti-NeuN (1 μg/ml; Millipore, Billerica, MA, USA) or rabbit anti-myeloperoxidase (1:200; Thermo scientific, Fremont, CA, USA) overnight. After washing, the sections were incubated with biotinylated anti-mouse or anti-rabbit IgG secondary antibody (20 μg/ml; GE Healthcare, UK) for 2 hours. Sections were processed with ABC reagents using a Vector ABC kit (Vector laboratories, Burlingame, CA, USA). The horseradish peroxidase reaction was detected with diaminobenzidine and H2O2. For immunofluorescence staining, the sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:500 for 2 hours at room temperature. Negative controls performed with secondary antibody alone showed no staining.
Assessment of neuronal survival
At 7 days and 8 weeks after hypoglycemia, rats were killed and their brains were harvested. The sections were immunostained with anti-NeuN. The number of NeuN-stained CA1 neurons was determined in every twelfth coronal section spanning the septal hippocampus (Bregma level −1.8 to −3.8) using unbiased stereology (Stereo Investigator, MicroBrightField, Williston, VT, USA). A counting frame (15 × 15 × 20 μm) was placed at the intersection of a matrix (200 × 200 μm) randomly superimposed onto the region of interest by the program.
Assessment of neuronal death
For identifying degenerating neurons in the hippocampus, Fluoro-Jade B (FJB) staining was performed as described previously . Briefly, sections were immersed in a basic alcohol solution for 5 minutes and 0.06% KMnO4 for 15 minutes, and then the sections were incubated in 0.0004% FJB (Histo-Chem, Jefferson, AR, USA) for 20 minutes. The slides were washed in distilled water, and then dried. To quantify neuronal death, every twelfth coronal section spanning the septal hippocampus (Bregma level −1.8 to −3.8) was analyzed from each animal. A blinded observer to the minocycline treatment condition counted the number of FJB positive (+) neurons in the hippocampal CA1 from both hemispheres. Mean counts of FJB (+) neurons from each region were used for statistical analysis.
Immunohistochemistry for evaluation of microglial activation
Scoring for microglial activation
Cell number (cell number per 200 μm × 200 μm)
Morphology (% with amoeboid morphology)
Assessment of infiltrated neutrophils
At 3 days after hypoglycemia, rats were killed and their brains were harvested. The sections were immunostained with anti-myeloperoxidase (anti-MPO). A blinded observer counted the number of MPO-immunopositive cells in the cortex and hippocampus of both hemispheres. Three sections from each animal were evaluated.
Six weeks following the sham operation or hypoglycemia, rats were first tested in the novel open field to evaluate the effects of the delayed treatment with minocycline on locomotor activity and exploratory behavior following hypoglycemia. They were then assessed by the Barnes maze test to evaluate spatial learning and memory . In the open field test, rats were placed in a brightly lit, square plexiglas enclosure (40 × 40 inches) surrounded by automated infrared photocells interfaced with a computer (Hamilton Kinder, San Diego, CA, USA) to record the data. Beam-breaks generated by movement were monitored, allowing measurements of the number of movements, distance moved, and time spent in the enclosure or zones. On each of three consecutive days, open field activity was recorded for 10 minutes after an initial 1-minute adaptation period. For analysis of the exploratory behavior, the arena was divided on a zone map consisting of a center region (15 × 15 inches2), four corner regions of 7.5 × 7.5 inches each, and a peripheral region (the remaining area). In the Barnes maze, a black acrylic escape tunnel was placed under one of the holes on a circular platform (120 cm in diameter) with 18 holes (10 cm in diameter per hole) along the platform perimeter (Hamilton Kinder). The platform was elevated 60 cm above the floor. Rats from each treatment group were randomly assigned to locate the escape tunnel from one of the three pre-determined locations to rule out spatial preference. Mildly noxious stimuli, blowing fans and 500 LUX of bright light, were used to increase the incentive in finding the escape tunnel. The Noldus EthoVision video tracking system (Noldus, Leesburg, VA, USA) was used to record and analyze the data. Rats were trained to locate the escape tunnel in two successive daily sessions for 5 days (three trials per session, three minutes per trial) with a one-hour intersession interval from different counterbalanced starting positions.
Data are shown as means ± standard error of the mean (SEM). Statistical significance was assessed by analysis of variance (ANOVA) and post hoc testing was accomplished using the Bonferroni test. P-values less than 0.05 were considered statistically significant. Behavioral data were evaluated by two-way repeated measures ANOVA (RANOVA) followed by post hoc pair-wise comparisons using the Bonferroni test. Microglial activation data were assessed by Kruskal-Wallis non-parametric one-way ANOVA followed by Dunn’s test for multiple group comparison.
Hypoglycemic brain injury induces microglial activation in the brain
Minocycline inhibits microglial activation after hypoglycemia
Minocycline reduces hippocampal neuronal death after hypoglycemia
Minocycline reduces MPO immunoreactivity induced by hypoglycemia
Minocycline reduces motor hyperactivity caused by hypoglycemia
Minocycline reduces spatial learning and memory impairment caused by hypoglycemia
Rats were subjected to memory testing using spatial learning in the Barnes maze, a test that relies heavily on hippocampal function. Over the five days of training, all groups learned the spatial task as evidenced by a progressive reduction in the distance traveled to reach the escape tunnel in the Barnes maze (F4, 200 = 76.1, P < 0.0001). Two-way RANOVA revealed a significant effect of hypoglycemia on spatial learning during Barnes maze acquisition (hypoglycemia effect: F1,50 = 5.98, P < 0.05). Although minocycline did not have an overall effect on learning (minocycline effect: F1,50 = 1.21, P > 0.27), it was able to lessen the deficit observed in the hypoglycemic (P < 0.05, post hoc test) but not in sham animals (P > 0.1, post hoc test). Due to the differential effect of minocycline on euglycemic and hypoglycemic animals, there was a significant minocycline-hypoglycemia interaction (F1,50 = 7.24, P < 0.01) (Figure 6C).
Minocycline has a long-term protective effect on neuronal death after hypoglycemia
The present study shows that delayed treatment with minocycline reduces microglial activation and neuronal death in the hippocampus after hypoglycemia. Minocycline treatment also prevented cognitive impairment at later time points. These results suggest that minocycline is an effective therapeutic candidate to prevent neuronal death and subsequent cognitive impairment after hypoglycemia.
The mechanisms of hypoglycemia-induced neuronal injury represent a more complex process than a simple lack of glucose supply to the brain [32, 33]. Rather, several contributing factors are involved in downstream events of hypoglycemia-induced neuronal death such as sustained activation of glutamate receptors , poly(ADP-ribose) polymerase activation , zinc translocation  and NADPH oxidase activation . Although several studies have been reported to intervene in this neuronal death cascade, there is currently no clinically applicable intervention strategy available. Therefore, this study has sought an indirect intervention strategy for preventing hypoglycemia-induced neuronal death and cognitive impairment that may be more clinically relevant.
The brain injury produced by hypoglycemia maturates over a period of several hours or days as seen in ischemia . Especially in hypoglycemia, delayed hippocampal damage is observed 3 to 5 days after the insult in CA1 pyramidal neurons , suggesting that mechanisms that develop slowly after hypoglycemia have a role in hypoglycemic neuronal death. Recent studies have shown that inflammatory cells infiltrate the ischemic area  or the hypoglycemic brain area . Inflammation is now recognized as a significant contributing mechanism in cerebral ischemia because anti-inflammatory compounds or inhibitors of iNOS and cyclooxygenase-2 reduce ischemic damage and improve the outcome of animals after ischemic insult [37, 38].
Microglial activation contributes to ischemia and traumatic brain injury-induced neuronal death. Previously our lab showed microglia are activated after hypoglycemia . Microglia are the major innate immune cells resident in the brain. Once activated by neurological damage or systemic inflammatory events, microglia release neurotoxic substances such as nitric oxide, ROS, cytokines, and chemokines, and undergo morphological change from a ramified to an amoeboid shape [13, 14]. Whether microglial activation is a net harmful or beneficial process is still controversial , however, there is evidence that indicates that early phage inflammation by acute brain injury can contribute to neuronal death. Our previous study shows that hypoglycemia induces microglial activation in the brain, which is affected by body temperature and vesicular zinc release . However, it is unknown whether the prevention of microglial activation by minocycline after hypoglycemia is neuroprotective.
Infiltrating peripheral inflammatory cells play important roles in the development of pathophysiological response following neurological damage. Brain damage, such as ischemic or traumatic injury, triggers physiological changes and neuronal death that induces adhesion of circulating leukocytes, leading to their migration into brain, causing release of pro-inflammatory substances [17, 40]. MPO produced from neutrophils has been used as a marker of infiltrating neutrophils and is involved in brain damage following such events as traumatic brain injury and cerebral ischemia. Numerous studies have been reported in which the accumulation of MPO-positive neutrophils into the ischemic brain is correlated with ischemic brain damage, although MPO gene deletion has been shown to exacerbate brain injury, which is mediated by the peroxynitrite reaction but not MPO [31, 41]. Activation of microglia precedes neutrophil infiltration and seems to play a role in the recruitment of neutrophils following brain damage . In our study, the MPO-positive neutrophils were observed in the hippocampal formation following hypoglycemic brain injury and this recruitment was prevented by minocycline. It suggests that neutrophil infiltration may be involved in brain inflammation and neuronal death after hypoglycemic injury and may be prevented by minocycline treatment.
Although minocycline was developed as an anti-microbial drug for the treatment of various infectious diseases, many other functions such as anti-apoptotic and anti-inflammatory effects have been identified . In particular the anti-inflammatory properties of minocycline have been observed in acute and chronic neurological disease animal models, as well as in human clinical trial studies [22, 23, 44]. In an animal model of multiple sclerosis, minocycline decreased the transmigration of T lymphocytes and inhibited the activation of metalloproteinases that degrade the extracellular matrix proteins of the basal lamina surrounding blood vessels, causing neutrophil infiltration [45, 46]. Based on these studies, our present results suggest that delayed treatment with minocycline can have a neuroprotective effect on hypoglycemic neuronal death by inhibiting microglial activation and neutrophil infiltration.
Since most hypoglycemic patients visit the emergency room several hours after the hypoglycemic episode, we delivered the initial dose of minocycline 6 hours after hypoglycemic insult. Microglial activation was detected in the hippocampus 24 hours after hypoglycemia. Thus we believe that treatment of 6-hours post-hypogecemic insult is a reasonable therapeutic window. Although we injected minocycline from 6 hours after hypoglycemia, microglial activation was significantly reduced, as was neuronal death. These results suggest that delayed treatment with minocycline suppressed microglial activation, which may decrease release of toxic substances such as nitric oxide, and IL6, etcetera. This further suggests that acute microglial activation after hypoglycemia is detrimental to neuronal survival.
Since minocycline is one of the most lipid-soluble tetracycline-class antibiotics, it can easily penetrate into the brain. Minocycline also has a long half-life compared to other tetracycline antibiotics . Thus, one or two doses of 50 to 100 mg per day of minocycline are effective in many patients to treat bacterial infection. A recent clinical study found that patients who received 200 mg of minocycline for five days within 24 hours after ischemic stroke showed significantly better outcome compared with patients receiving placebo . In the present study, we used 50 mg/kg per day for one week in rats. We understand that this concentration of minocycline is fairly high for a single dose. Therefore, for clinical applications, a single dose of minocycline for prevention of hypoglycemia-induced neuronal death should be re-evaluated.
Learning and memory deficits are common neurological sequelae following hypoglycemia in patients with type 1 diabetes and in the relatively younger population with type 2 diabetes [49–51]. Our previous study showed that hypoglycemia-induced hippocampal damage induced impairment of learning and memory . In the present study, the Barnes maze test was performed to evaluate spatial learning and memory. As seen in our previous study using the water maze test, subjects experiencing severe hypoglycemia displayed a longer distance traveled to reach the escape tunnel, implying that spatial learning has been compromised. However, minocycline treatment reduced the travel distance in rats who experienced hypoglycemia. It has been reported that minocycline improves cognitive impairment in focal cerebral ischemia , Alzheimer’s disease models , and other animal models of neurological disease [54, 55].
Because tetracycline derivatives, like minocycline, have anti-inflammatory properties and are clinically well tolerated, we studied whether minocycline could serve as a neuroprotective compound against hypoglycemia-induced brain injury. In the present study, we report that in a rat model of hypoglycemia, 1) minocycline is neuroprotective, even when the treatment is initiated 6 hours after hypoglycemia; 2) minocycline prevents microglial activation after hypoglycemia; and 3) minocycline prevents cognitive impairment even at several weeks after hypoglycemia. Thus, the present study suggests that prevention of microglial activation by minocycline has a strong therapeutic potential for prevention of hypoglycemia-induced brain injury.
The authors declare no competing interests.
This work was supported by the Department of Veterans Affairs (JL) and by the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120202) to SWS.
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