Small shifts in craniotomy position in the lateral fluid percussion injury model are associated with differential lesion development.

Previous studies have shown that location and direction of injury may affect outcome in experimental models of traumatic brain injury. Significant variability in outcome data has also been noted in studies using the lateral fluid percussion brain injury model (FPI) in rats. In recent studies from our laboratory, we observed considerable variability in localization and severity of tissue damage as a function of small changes in craniotomy position. To further address this issue, we examined the relationship between craniotomy position and brain lesion size/location in rats subjected to moderate FPI (2.28 +/- 0.18 atmospheres). With placement of a 5-mm craniotomy adjacent to the sagittal suture, there was both ipsilateral and contralateral damage as detected at 3 weeks posttrauma using T2-weighted magnetic resonance imaging (MRI). The MRI lesions were generally restricted to the hippocampus and subcortical layers. Shifting of the craniotomy site laterally was associated with increased ipsilateral tissue damage and a greater cortical component that correlated with distance from the sagittal suture. In contrast, the contralateral MRI lesion did not change significantly in size or location unless the center of the craniotomy was placed more than 3.5 mm from the sagittal suture, under which condition contralateral damage could no longer be detected. Ipsilateral tissue damage as determined from the MRI scans was linearly correlated to motor outcome but not with cognitive outcome as assessed by the Morris Water Maze. We conclude that craniotomy position is critical in determining extent and location of tissue injury produced during the lateral FPI model in rats. Addressing such potential variability is essential for studies that address either injury mechanisms or therapeutic treatments.

Subtle alterations in NMDA-stimulated cyclic GMP levels following lateral fluid percussion brain injury.

This study examined whether NMDA-stimulated cyclic GMP levels were altered at two different time points following lateral fluid percussion injury. At 60 min and 15 days postinjury, the left and right hippocampi were dissected and chopped into mini-prisms. Each hippocampus was divided into five equal parts and incubated with either the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine, 500 microM) alone, IBMX and N-methyl-D-aspartic acid (NMDA) OR IBMX, NMDA, and glycine (10 MM). Two concentrations of NMDA were used: 500 or 1,000 microM. Tissues were then assayed for levels of cyclic GMP. Results indicated that there were no changes in basal levels of cyclic GMP at either postinjury time point. At 60 min postinjury, there were no significant main effects for injury or drug concentration. There was a significant injury x side interaction effect with increased levels of NMDA-stimulated cyclic GMP in the hippocampus ipsilateral to the injury impact and decreased cyclic GMP levels in the contralateral hippocampus. There were no significant alterations in NMDA-stimulated cyclic GMP levels at 15 days postinjury. The data from this study indicated that NMDA-stimulated cyclic GMP accumulation is differentially altered in the hippocampus ipsilateral and contralateral to the site of the injury at 1 h after injury, but is normalized by 15 days postinjury. These findings implicate NMDA-mediated intracellular signaling processes in the acute excitotoxic response to injury.

Interaction of cisplatin and DNA-targeted 9-aminoacridine platinum complexes with DNA.

Interaction of acridine- and 9-aminoacridinecarboxamide platinum complexes with DNA was investigated with respect to their DNA sequence specificity and kinetics of binding. The DNA sequence specificity of the compounds was quantitatively analyzed using a polymerase stop assay with the plasmid pUC19. The 9-aminoacridinecarboxamide platinum complexes exhibited a different sequence specificity to that of cisplatin, shifted away from runs of consecutive guanines (the main binding site for cisplatin). This alteration was dependent on chain length. Shorter chain length compounds (n = 2, 3) showed a greater difference in sequence specificity, while longer chain length compounds (n = 4, 5) more closely resembled cisplatin. An acridinecarboxamide platinum complex showed a similar sequence specificity to cisplatin, revealing that the major change of sequence specificity was due to the presence of the 9-amino substituent. A linear amplification system was used to investigate the time course of the reaction. The presence of an intercalating group (acridinecarboxamide or 9-aminoacridinecarboxamide) greatly increased the rate of reaction with DNA; this is proposed to be due to a different reaction mechanism with DNA (direct displacement by the N-7 of guanine).

Protein-DNA footprinting of the human epsilon-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents.

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein-DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy epsilon-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences - both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein-DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5'- and 3'-ends of the CCAAT box in K562 cells - this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the epsilonF1 DNA binding motif in K562 cells.

1H NMR study of robustoxin, the lethal neurotoxin from the funnel web spider Atrax robustus.

Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.

Protein-DNA interactions in the human beta-globin locus control region hypersensitive site-2 as revealed by four nitrogen mustards.

Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other sites of protection and enhancement that corresponded to known transcription factor binding sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.

Effect of tetrahydroaminoacridine, a cholinesterase inhibitor, on cognitive performance following experimental brain injury.

An emerging literature exists in support of deficits in cholinergic neurotransmission days to weeks following experimental traumatic brain injury (TBI). In addition, novel cholinomimetic therapeutics have been demonstrated to improve cognitive outcome following TBI in rats. We examined the effects of repeated postinjury administration of a cholinesterase inhibitor, tetrahydroaminoacridine (THA), on cognitive performance following experimental TBI. Rats were either injured at a moderate level of central fluid percussion TBI (2.1+/-0.1 atm) or were surgically prepared but not delivered a fluid pulse (sham injury). Beginning 24 h after TBI or sham injury, rats were injected (IP) daily for 15 days with an equal volume (1.0 ml/kg) of either 0.0, 1.0, 3.0, or 9.0 mg/kg THA (TBI: n = 8, 8, 10, and 7, respectively, and Sham: n = 5, 7, 8, 7, respectively). Cognitive performance was assessed on Days 11-15 after injury in a Morris water maze (MWM). Analysis of maze latencies over days indicated that chronic administration of THA produced a dose-related impairment in MWM performance in both the injured and sham groups, with the 9.0 mg/kg dose producing the largest deficit. The 1.0 and 3.0 mg/kg doses of THA impaired MWM performance without affecting swimming speeds. Thus, the results of this investigation do not support the use of THA as a cholinomimetic therapeutic for the treatment of cognitive deficits following TBI.

Interaction of 11 cisplatin analogues with DNA: characteristic pattern of damage with monofunctional analogues.

In this paper the sequence specificity of DNA damage has been determined for 11 cisplatin analogues. A number of the analogues used in this study have been included in clinical trials. A Taq DNA polymerase linear amplification technique was utilised to ascertain the sequence selectivity of cisplatin analogues damage to DNA. The analogues differed in their ability to damage DNA with cisplatin being the most effective DNA damaging agent followed by (in decreasing order): tetraplatin (tetrachloro(1,2-diaminocyclohexane)platinum(IV) (RR isomer)), cis-dichlorobis(isopropylamine)platinum(II), dichloro(1,2-diaminocyclohexane)platinum(II) (SS isomer), dichloro(1,2-diaminocyclohexane)platinum(II) (RR isomer), cis-bis(cyclohexylamine)dichloroplatinum(II), carboplatin, cis-dichlorobis(isopentylamine)platinum(II), and CHIP (cis-dichloro-trans-dihydroxybis(isopropylamine)platinum(IV)). However, the sequence specificity of these analogues was similar in position and relative intensity of damage. We also provide evidence that platinum(IV) complexes can damage DNA without being reduced to platinum(II). It was found that a 10-fold higher concentration of cisplatin was required to damage DNA in Tris-HCl compared to Hepes buffers. In this paper we have detected a characteristic pattern of damage with monofunctional analogues that could be used to determine the mode of binding of a cisplatin analogue with DNA. The monofunctional analogues tested were chloro(diethylenetriamine)platinum(II) and cis-diamminechloro(1-octylamine)platinum(II) as well as transplatin.

Chronic, post-injury administration of D-cycloserine, an NMDA partial agonist, enhances cognitive performance following experimental brain injury.

The purpose of this study was to determine the effect of augmenting NMDA receptor activation on cognitive deficits produced by traumatic brain injury (TBI). Specifically, D-cycloserine (DCS), a partial agonist of the NMDA-associated glycine site, was tested as a potential cognitive enhancer. Rats were injured using lateral fluid percussion TBI (2.8 +/- .10 atm). On days 1-15 post-injury, animals were injected (i.p.) with vehicle (n = 8), 10 mg/kg (n = 9), or 30 mg/kg (n = 8) of DCS. Sham-injured animals treated with either vehicle (n = 8) or 30 mg/kg of DCS (n = 8) were used for comparison. On days 11-15 post-injury, cognitive function was assessed using the Morris water maze (MWM). Results indicate that the 30 mg/kg dose of DCS significantly attenuated memory deficits as compared to injured vehicle-treated animals (P < 0.01). Analysis also revealed that performance of the injured-DCS (30 mg/kg) group was not significantly different from sham-injured animals treated with vehicle (P > 0.10). In contrast, the 10 mg/kg dose of DCS was ineffective in reducing injury-induced memory deficits. DCS (30 mg/kg) also significantly improved the spatial memory of sham-injured animals when compared with sham-injured animals treated with vehicle (P < 0.05). In conclusion, chronic, post-injury enhancement of the NMDA receptor is an effective strategy for ameliorating TBI-associated cognitive deficits.

Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer peptide modified by O-linked N-acetylglucosamine.

Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.

Working memory deficits following traumatic brain injury in the rat.

This study was designed to examine working memory following fluid-percussion traumatic brain injury (TBI) using the Morris water maze (MWM). Rats were injured (n = 9) at a moderate level of central fluid percussion injury (2.1 atm) or were prepared for injury but did not receive a fluid pulse (sham injury) (n = 10). On days 11-15 postinjury, working memory was assessed using the MWM. Each animal received 8 pairs of trials per day. For each pair of trials, animals were randomly assigned to one of four possible starting points and one of four possible escape platform positions. On the first trial of each pair, rats were placed in the maze facing the wall and were given 120 sec to locate the hidden escape platform. After remaining on the goal platform for 10 sec, they were placed back into the maze for the second trial of the pair. The platform position and the start position remained unchanged on this trial. After the second trial, the animal was given a 4 min intertrial rest. Between pairs of trials, both the start position and the goal location were changed. Analyses of the latency to reach the goal platform indicated that sham-injured animals performed significantly better on the second trial than on the first trial of each pair. However, injured animals did not significantly differ between first and second trial goal latencies on any day. These results indicate that injured animals have a profound and enduring deficit in spatial working memory function on days 11-15 after TBI.

The effect of postinjury administration of polyethylene glycol-conjugated superoxide dismutase (pegorgotein, Dismutec) or lidocaine on behavioral function following fluid-percussion brain injury in rats.

Previous studies in our laboratory have shown that polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) or lidocaine treatment before experimental fluid-percussion brain injury in rats reduces the cortical hypoperfusion normally found in the early posttraumatic period. The purpose of the current study was to determine if posttreatment with PEG-SOD or lidocaine is also associated with changes in the trauma-induced suppression of motor and cognitive function that occurs following traumatic brain injury (TBI). Twenty-four hours after surgical preparation, rats were randomly assigned to a saline or drug posttreatment group, PEG-SOD (pegorgotein, Dismutec 10,000 IU/kg) or lidocaine (2 mg/kg), which was injected iv 30 min after moderate injury. PEG-SOD completely prevented beam walk deficits on days 1-5 postinjury while lidocaine similarly prevented beam walk deficits on days 2 through 5 postinjury. Both drugs produced a statistically insignificant trend for a decrease in beam balance duration deficits on days 1-5 postinjury and had no effect on cognitive function, as assessed by the Morris water maze, on days 11 through 15 postinjury. The mechanism by which PEG-SOD and lidocaine reduce posttraumatic motor deficits may be related to their free radical scavenging effect or previously reported effects on posttraumatic cerebral blood flow. To our knowledge, this is the first report of the effectiveness of these two agents in laboratory animals when administered after traumatic injury.

Reduced sensorimotor reactivity following traumatic brain injury in rats.

The present study examined sensorimotor reactivity in rats following traumatic brain injury (TBI). Moderate injury was induced with midline fluid percussion in some of the rats. Others received identical surgery, but were not injured (sham-injured rats), or received neither surgery nor injury (naive rats). All rats were evaluated in acoustic and/or tactile startle procedures. At 8 days post-injury, the sensorimotor reactivity of TBI rats to acoustic stimuli was severely reduced compared to that of sham-injured rats. This TBI-induced deficit was enduring (> 30 days). In a separate experiment, greater sensorimotor reactivity was observed with tactile (vs. acoustic) stimulation in both TBI and naive rats although startle amplitudes for the TBI rats were lower than control levels for both types of stimuli. These results suggest that sensorimotor reactivity is altered by TBI and that the startle procedure is a promising method for investigation of information processing alterations following TBI.

Exposure to environmental complexity promotes recovery of cognitive function after traumatic brain injury.

This study was designed to determine whether exposure to a complex environment after traumatic brain injury (TBI) would promote the recovery of cognitive function. Rats were injured at a moderate level of fluid percussion injury (2.1 atm) or were prepared for injury but were not injured (sham injury). Immediately after the injury or sham injury, the injured/complex (n = 8) and the sham/complex (n = 7) groups were placed into a complex environment. The complex environment was a 89 x 89-cm enclosure with different types of bedding and objects that provided motor, olfactory, tactile, and visual stimulation. The injured/standard (n = 8) and the sham/standard (n = 8) groups were returned to the animal vivarium where they were housed individually in standard wire mesh cages (24 x 20 x 18 cm). On days 11-15 (postinjury), performance in the Morris water maze was assessed. Analysis of the latency to reach the goal platform indicated that injured animals recuperating in the complex environment performed significantly better than injured animals recovering in the standard environment (p < 0.01). In fact, injured animals in the complex environment performed as well as both sham-injured groups. The improved performance of injured rats recovering in the enriched environment occurred in the absence of environmentally induced alterations in brain weight. These results indicate that exposure to environmental complexity enhances recovery of cognitive function after TBI.

The effect of postinjury kindled seizures on cognitive performance of traumatically brain-injured rats.

The purpose of this experiment was to examine the consequences of postinjury seizures on cognitive performance after experimental traumatic brain injury (TBI). Rats either were injured at a moderate (2.1 atm) level of central fluid percussion TBI (n = 16) or were surgically prepared but did not receive a fluid pulse (sham-injured control, n = 16). Beginning 24 h after TBI, injured animals were injected (ip) once daily (Days 1-24 postinjury) with either saline (n = 8) or 25 mg/kg pentylenetetrazol (PTZ) (n = 8). Sham-injured rats were injected with an equal volume of saline (n = 8) or PTZ (n = 8). In both injured and sham-injured animals, daily injections of PTZ resulted in an increase in the severity of behavioral seizures over days. On Days 25-29 after injury or sham injury, all animals were tested in the Morris water maze (MWM). Analysis of maze performance indicated that in sham-injured animals PTZ-produced seizures had a detrimental effect on performance. In injured animals, however, PTZ-treated animals exhibited significantly faster acquisition and better terminal performance in the MWM than did untreated injured animals. These results show that posttraumatic kindled seizures do not exacerbate behavioral deficits after TBI and may, in fact, improve recovery following injury. The findings of this experiment are consistent with the hypothesis that post-TBI neuronal depression may contribute to behavioral morbidity following injury.

Impaired gustatory neophobia following traumatic brain injury in rats.

To investigate the function of the amygdala following traumatic brain injury (TBI), rats were tested on a gustatory neophobia task that is sensitive to amygdala and hippocampal damage. Rats were either injured at a moderate level of fluid percussion injury (2.1 atm) or surgically prepared but not injured (sham-injury). Seven days after injury (n = 8) or sham injury (n = 9), rats were habituated to the testing chamber without food items present for 30 min. All rats were then food deprived. Twenty-four hours later, rats were placed in the testing chamber for 30 min and allowed to eat freely from four dishes of different foods: rat chow, raisins, potatoes, and cookies. Results showed that injured and sham-injured rats did not differ in their ability to find hidden food, suggesting that TBI does not produce an enduring impairment of olfaction. There was also no difference in the total amount of food eaten between injured and sham groups (p > 0.05). The percentage of each type of food consumed did differ between the two groups with sham controls consuming more familiar food (rat chow) compared to the unfamiliar foods (p < 0.01). The injured animals distributed their eating evenly among the four foods with no particular preference for any one food (p < 0.05). This pattern of eating behavior in injured animals is similar to animals that have lesions to both the hippocampus and amygdala (Sutherland and McDonald, 1990). Therefore, the results of this experiment suggest that, in addition to the hippocampus, the amygdala may also contribute to the behavioral changes observed following TBI.