Secondary growth of a cortical necrosis: effect of NOS inhibition by aminoguanidine post insult.

BACKGROUND: A cortical tissue necrosis from a focal freezing injury expands to 140% of its initial volume within 24 hrs in rats. Previous studies of our laboratory have shown that administration of the NOS inhibitor aminoguanidine (AG) prior to trauma attenuates this process of secondary brain damage. Objective of the present study was to analyse whether this agent is also protective when treatment commences after the insult. METHOD: A highly standardized freezing lesion was induced in the brain cortex of 30 anaesthetized rats. The animals were divided into three experimental groups. Animals of group I (sham-5 min, n=10) were sacrificed 5 min after trauma for quantitative histomorphometric assessment of the primary cortical lesion. Animals of group II (sham-24 h, n=10) received isotonic saline (16.7 ml/kg b.w., i.p.) at 15 min and 8 hrs after trauma. In the treatment group (group III, AG-24 h, n=10), AG was administered (100 mg/kg b.w.) also at 15 min and 8 hrs after trauma. 24 hrs later--the time point of maximal lesion spread--the animals of group II and III were sacrificed for quantification of the secondary lesion growth. FINDINGS: The focal freezing injury produced a cortical necrosis volume of 6.07+/-1.04 mm(3) immediately after trauma (group I). After sham treatment, the necrosis expanded to 8.39+/-1.57 mm(3) within 24 hrs (group II) corresponding to a lesion growth of 138% compared to the primary necrosis ( p<0.01 vs. group I). In animals treated with AG after the trauma (group III), the volume of necrosis was significantly attenuated at 24 hrs to 6.77+/-0.87 mm(3) representing an expansion of the lesion to only 112% ( p<0.05 vs. group II). Thus, AG was inhibiting the secondary growth of necrosis by no less than 69%. INTERPRETATION: The findings demonstrate that AG retains its neuroprotective potential against secondary brain damage from trauma even when administration begins after trauma.

Role of nitric oxide in the secondary expansion of a cortical brain lesion from cold injury.

We have investigated the role of nitric oxide (NO) as mediator of the secondary growth of a traumatic cortical necrosis. For this purpose, a highly standardized focal lesion of the brain was induced in 46 Sprague-Dawley rats by cold injury. Twenty-four hours later--the timepoint of maximal lesion spread--the animals were sacrificed and brains were removed for histomorphometry of the maximal necrosis area and volume. The animals were divided into five experimental groups. Group I received the NO donor L-arginine as i.v. bolus 10 min prior to trauma (300 mg/kg body weight; n = 10) and a second bolus of the same dosage intraperitoneally 1 h after trauma. Group II (n = 10)--serving as control of group I--was infused with an i.v. bolus of 1 mL/kg isotonic saline 10 min prior to and a subsequent bolus i.p. 1 h after trauma. Group III (n = 8) received 100 mg/kg b.w. of the inducible NOS (iNOS) inhibitor aminoguanidine (AG) 1 h before and 8 h after trauma by intraperitoneal route. Group IV was administered with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NNA; 100 mg/kg b.w., i.p.; n = 8); group V--the controls of group III and IV--was administered with isotonic saline (1 mL/kg b.w. i.p.; n = 10) 1 h before and 8 h after trauma. In the control group with i.v./i.p. sham treatment (II), the focal lesion led to a cortical necrosis with a maximum area of 3.1 +/- 0.3 mm2 and a lesion volume of 5.7 +/- 0.5 mm3 at 24 h after trauma. In animals with administration of L-arginine, the focal lesion had a maximum area of 3.1 +/- 0.3 mm2 and a volume of 5.3 +/- 0.5 mm3. Hence, the NO donor did not affect the secondary growth of necrosis. Animals with i.p. sham treatment (group V) had a maximal lesion area of 3.6 +/- 0.2 mm2 and lesion volume of 6.2 +/- 0.4 mm3. Administration of aminoguanidine afforded significant attenuation of the lesion growth. Accordingly, the maximal area of necrosis spread only to 2.8 +/- 0.2 mm2 with a volume of 4.5 +/- 0.5 mm3, respectively, at 24 h after trauma (p < 0.01 vs group V). On the other hand, administration of L-NNA did not influence the maximal lesion area (3.7 +/- 0.2 mm2) or lesion volume (6.5 +/- 0.5 mm3) evolving at 24 h after trauma. Thus, neither the enhancement of the formation of NO by L-arginine nor gross inhibition of the synthesis of NO by L-NNA did affect the secondary spread of the necrosis from a focal trauma. The marked attenuation of the posttraumatic necrosis growth by the iNOS inhibitor aminoguanidine strongly indicates an important role of iNOS product in this phenomenon. These findings, thus, demonstrate that the expansion of a primary necrotic focal lesion is a secondary process which can be therapeutically inhibited. Thereby, the growth of a focal tissue necrosis from trauma is clearly identified as a manifestation of secondary brain damage. This information is deemed important for the better understanding of the pathophysiology of traumatic brain injury and for the targeted development of specific treatment modalities.

Interstitial lactate in the peritraumatic penumbra of rat brain.

A traumatic brain tissue necrosis is expanding to approximately 150% within 24 h after lesion. This process is accompanied by marked reduction of the perifocal cerebral blood flow likely to activate anaerobic glycolysis from a reduced O2-supply leading to an accumulation of lactic acid. The current study was carried out to assess the interstitial levels of lactic acid as a potential factor of secondary brain damage. A microdialysis probe was stereotactically implanted approximately 2 mm below the brain surface of the parietal cortex in Sprague Dawley rats (250-300 g bw; n = 6) in chloralhydrate anaesthesia. The position of the probe was controlled by histology. 24 h later a standardised cortical cold injury was induced above the probe in halothane/N2O anaesthesia. Dialysate (2 microliters/min) was collected in 15 min intervals, starting 1 h prior to and continuing until 4 hrs after trauma. The lactate concentration in the dialysate was fluorometrically determined by an enzymatic assay. Under baseline conditions dialysate concentrations of 324 +/- 48 microM were observed. A release of lactate was not found initially after trauma. Between 70 and 105 min later, however, the interstitial lactate levels briefly increased to 416 +/- 34 microM (n.s.), while reaching baseline levels again thereafter. Thus, the current results do not confirm an increased accumulation of lactate in the interstitial compartment of the penumbra despite a marked perifocal hypoperfusion of the brain after focal injury. The transitory increase in lactate at 90 min after trauma is unlikely to have caused a severe tissue acidosis which might be held liable for the secondary growth of the brain lesion induced by the focal injury.

Attenuation of secondary lesion growth in the brain after trauma by selective inhibition of the inducible NO-synthase.

In previous studies we have demonstrate that aminoguanidine pretreatment attenuates the secondary necrosis growth after focal brain trauma. Purpose of the present investigation was to elucidate the therapeutic potential of this iNOS-inhibitor when administered post lesion. Sprague-Dawley rats were subjected to a highly standardized cortical freezing lesion and administered with aminoguanidine (100 mg/kg i.p.) 15 min and 8 hrs after trauma or with isotonic saline, respectively. Animals were assigned to one of three experimental groups. The animals of group I--which served as reference for the histomorphometric determination of the spread of the primary lesion--were sacrificed 5 min after trauma. Group II, receiving isotonic saline and group III with aminoguanidine were subjected to perfusion fixation 24 hrs after trauma for evaluation of the necrosis growth. In controls with saline, the volume of the cortical necrosis increased from 6.07 +/- 1.04 mm3 (5 min) to 8.39 +/- 1.57 mm3 at 24 hrs (group II) after trauma. Treatment with aminoguanidine (group III) led to significant attenuation of the expansion of the necrosis to 6.77 +/- 0.87 mm3 at 24 hrs. Thus, the pathological role of activation of the inducible NO-synthase in the phenomenon of secondary lesion growth is confirmed by the present data on iNOS-inhibition. Attenuation of expansion of the lesion is achieved even when initiating therapy after trauma.