alterations in hippocampal synaptic plasticity may be responsible for learning and memory deficits after TBI.
was not induced. Normal body temperature was maintained throughout the whole period of anesthesia.
Recent data from diverse brain injury models, includ- ing closed head injury (CHI), middle cerebral artery occlusion, retinal exposure to glutamate, and endo- toxin (LPS)-induced neuroinflammation, demonstrate a loss of NMDAR, which is evident a few hours after injury and lasts 24 h or more (20–25). Thus, the time of onset and the duration of NMDAR hypofunction, rather than hyperfunction, are important factors deter- mining the outcome of NMDAR blockade. These ob- servations have prompted us to explore the effect of stimulation of NMDAR with the full agonist NMDA at 1 and 2 d after CHI. Indeed, we found that this treatment paradigm resulted in a progressive and significant amelioration of neurological and cognitive deficits, measured 14 d post-injury (20). Thus, our results fully support the notion that NMDAR activation may prove beneficial to TBI patients.
The present study had two major goals: first, to determine whether CHI leads to impaired LTP and whether it is associated with decline cognitive func- tions; second, to investigate the effect of D-cycloserine (DCS), a partial agonist of the NMDAR-associated glycine site, on LTP, motor and cognitive function, and neuronal cell death. DCS was selected because it has been shown to improve cognitive function in various animal models (26–29). DCS has a good safety profile, and it is already in use in humans in several different indications (30–33). These properties could signifi- cantly facilitate the clinical application of DCS to human TBI patients.
MATERIALS AND METHODS
Animals and DCS treatment
Male Sabra mice, 8–10 wk old, (strain of the Hebrew Univer- sity) were used in the study. Animals were maintained and treated according to the regulations of the Animal Care Committee of the Hebrew University. Food and water were provided ad libitum. D-cycloserine (Sigma, St. Louis, MO, USA) was dissolved in saline and intraperitoneally (i.p.) injected (10 mg/kg, 100 l/10gr) 24 h post-injury. Vehicle (saline) was i.p. injected in control CHI mice.
CHI was induced under ether anesthesia, confirmed by loss of pupillary and corneal reflexes, using a weight-drop device as described previously (12, 34, 35). Briefly, after induction of isoflurane anesthesia, a midline longitudinal incision was performed, the skin was retracted, and the skull was exposed. The left anterior frontal area was identified and a tipped TeflonTM cone was placed 1 mm lateral to the midline, in the midcoronal plane. The head was fixed and a 75 g weight was dropped on the cone from a height of 18 cm, resulting in a focal injury to the left hemisphere. After trauma, the mice received supporting oxygenation with 95% O2 for no longer than 2 min and were then brought back to their cages. Sham mice were anesthetized, their scalps were incised, but trauma
Mice were evaluated by a blinded examiner, using a set of 10 tasks, collectively called Neurological Severity Score (NSS, see Fig. 1A), which tests reflexes, alertness, coordination and motor abilities. One point is awarded for failure to perform a particular task; thus a normal mouse scores 0 (34). NSS was evaluated at 1h (NSS1h) to define severity of injury, and then every other day during 22 d. The extent of recovery was calculated as the difference between NSS1h and that at any other time, as shown: NSS NSS (1h) – NSS (24h).
Object recognition test (ORT) which tests memory function (36, 37) was performed by a blinded evaluator on days 3, 9 and 16 after CHI as described (20). Mice were placed in a test-cage with two identical objects and the cumulative time spent by the mouse at each of the objects was recorded during a 5 min interval. Four hours later, the mice were reintroduced into the cage, where one of the two objects was replaced by a new one, and the time spent at each of the objects was again recorded. The basic measure is the percent of the total time spent in exploring an object, whereby healthy rodents spend more time exploring a new object than a familiar, i.e., “memorized” object.
Coronal hippocampal slices (300–400 m) were prepared from sham, CHI-vehicle or CHI-DCS male Sabra mice 16 d following CHI. Slices were maintained for at least 2 h in artificial cerebrospinal fluid (aCSF) that contained 126 mM NaCl, 1.2 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 18 mM NaHCO3, and 11 mM glucose saturated with 95% O2, 5% CO2 at 25°C. After recovery, slices were sub- merged and continuously superfused with aCSF at 25°C. Field excitatory postsynaptic potentials (fEPSPs) were recorded from stratum-radiatum of the CA1 region with glass micro- electrodes filled with aCSF. Picrotoxin (100 M) was added to the bath solution to block GABAA receptor-mediated inhibi- tory postsynaptic potentials (fIPSPs). To evoke fEPSPs, Schaf- fer collateral/commissural afferents were stimulated with 0.1-Hz pulses using steel bipolar microelectrodes at intensities adjusted to produce an evoked response that was 40–50% that of the maximum-recorded fEPSP for each recording. Following stable baseline (from which the last 10 min are shown), LTP was induced by high frequency stimulation (100 Hz, 1 s duration, 2 trains at 10 s inter train intervals) at the same intensity as the test stimulus, and synaptic responses were monitored for 50 min after LTP. For measuring paired pulse ratio (PPR), paired stimuli were given with an inter- stimulus interval of 50 msec, and the ratio between the second and the first amplitude of the fEPSPs was calculated. Data were collected using Multiclamp 700B amplifier (Molec- ular Devices, CA), filtered at 2 kHz and digitized at 5–10 kHz. Compiled data were analyzed and expressed as the mean percent of fEPSP slope S.E.M over the base-line levels. Results in the text and figure are presented as the mean sem. Group comparisons were compared using a t test, either paired or unpaired as appropriate; P 0.05 was taken as indicating statistical significance.
The FASEB Journal
YAKA ET AL.