The radiographic appearance of the pulmonary vasculature in the cat.

The purpose of this study was to determine the dimensions of the pulmonary arteries and veins in the cat, both in normal animals and those with evidence of intrathoracic disease. The radiographs of 50 normal cats, 35 cats with hypertrophic cardiomyopathy (HCM), and 15 cats with other intrathoracic disease were assessed. The normal range for the right cranial lobar artery compared with the proximal third of the fourth rib was 0.5-1.0, with a mean artery-to-rib ratio of 0.70 (standard deviation +/- 0.13). Normal cats had a mean vein diameter of (0.20 +/- 0.03 cm) (P = 0.034) compared with cats with HCM, which had a significantly greater mean vein diameter (0.22 +/- 0.04 cm).

Modified-peptide inhibitors of amyloid beta-peptide polymerization.

Cellular toxicity resulting from nucleation-dependent polymerization of amyloid beta-peptide (Abeta) is considered to be a major and possibly the primary component of Alzheimer's disease (AD). Inhibition of Abeta polymerization has thus been identified as a target for the development of therapeutic agents for the treatment of AD. The intrinsic affinity of Abeta for itself suggested that Abeta-specific interactions could be adapted to the development of compounds that would bind to Abeta and prevent it from polymerizing. Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization. The activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents. Additional series of compounds prepared to probe structural requirements for activity allowed reduction of the size of the inhibitors and optimization of the Abeta-derived peptide portion to afford a lead compound, cholyl-Leu-Val-Phe-Phe-Ala-OH (PPI-368), with potent polymerization inhibitory activity but limited biochemical stability. The corresponding all-D-amino acyl analogue peptide acid (PPI-433) and amide (PPI-457) retained inhibitory activity and were both stable in monkey cerebrospinal fluid for 24 h.

Controlled modulation of BBB permeability using the bradykinin agonist, RMP-7.

Previous studies have shown that the bradykinin agonist, RMP-7, can safely permeabilize the blood brain barrier (BBB) by activation of constitutive B2 receptors on endothelial cells. The paper describes a series of studies using quantitative autoradiography and intracarotid infusions of RMP-7 to further elucidate the effect on BBB permeability. Because earlier studies also demonstrated that even greater effects of RMP-7 were observed in the BBB associated with brain tumors, animal models were employed so that comparisons could be made between the effects of RMP-7 within tumor, brain tissue proximal to tumor, and brain tissue distal from tumor. In the first study, the effect of RMP-7 on enhancing the BBB permeation of three compounds of different physical characteristics was directly compared ([14C]carboplatin, small, hydrophilic; [14C]dextran, large, hydrophilic; [14C]BCNU, small, lipophilic). RMP-7 increased permeability of the vascular barriers to both hydrophilic compounds, carboplatin and dextran. While the effects of RMP-7 were observed on nontumor BBB, the greatest and most consistent effects were observed on the blood brain tumor barrier. This was true for both carboplatin and dextran, with progressively less effect seen as the distance from tumor boundary increased. This topographic effect was more pronounced with the larger molecular weight compound, dextran. No effect of RMP-7 was seen in permeabilizing the BBB or the blood brain tumor barrier for the lipophilic drug, BCNU. In a second study, the generality of RMP-7's effects was established by demonstrating similar increases in permeability to carboplatin in both inbred (Fischer 344) and outbred (Wistar) rat strains, implanted with varying tumor cell lines. Finally, several additional studies were performed to gain greater insight into the dynamics involved with eventual restoration of the BBB following RMP-7 administration. In one series, it was demonstrated that the BBB begins to close nearly immediately upon withdrawal of RMP-7, with complete restoration occurring within minutes. In another series, tachyphylaxis or desensitization resulting from continuous RMP-7 infusion was studied. These studies demonstrated that 60 min of continuous RMP-7 infusion resulted in complete, spontaneous restoration of the barrier to both carboplatin and dextran. Moreover, the desensitization appears to be linked to the initial activation of the receptors in a way which suggests that obligatory desensitization may exist as part of a more complete response. These data are discussed as they relate to practical issues to enhance delivery of drugs across the BBB, as well as more fundamental issues involving the function of the BBB and its interaction with the brain.

Unlocking the blood-brain barrier: a role for RMP-7 in brain tumor therapy.

The effect and mechanism of the blood-brain barrier-permeabilizing agent, RMP-7, was investigated in a series of studies employing a rat RG2 glioma model. Changes in uptake of carboplatin into brain tumor and various nontumor brain tissue regions was determined using a sophisticated image analysis system. This system permitted quantitative autoradiography to be analyzed simultaneously with overlayed histological images from the same coronal brain section. A wide range of intracarotid doses of RMP-7 (0.01 to 9.0 micrograms/kg) was shown to significantly increase the permeability of carboplatin into tumor tissue and surrounding brain tissue (up to twofold) in a dose-dependent manner. Additionally, substantially greater permeability effects were seen in the tumor compared to healthy brain. Moreover, a clear topographic profile was observed in nontumor brain tissue, with progressively less uptake observed with increasing distance from the tumor. The fact that RMP-7 increased the uptake of carboplatin into ipsilateral brain tissue outside the tumor mass has potential implications for treating human glioma patients, for it is commonly recognized that tumor cells typically migrate from the tumor mass into surrounding brain tissue thereby escaping conventional attempts to destroy the malignant cells. To help elucidate the mechanism of RMP-7's permeability effects, the uptake of carboplatin was also determined under conditions where either the bradykinin B2 receptor antagonist, HOE140, or the B1 antagonist, [desArg10]HOE140, was coadministered with RMP-7. Results indicate that RMP-7's effects are mediated specifically through bradykinin B2 receptors. Furthermore, neither bradykinin antagonist alone affected the uptake of carboplatin into the leaky tumor region, suggesting that abnormal elevations in endogenous bradykinin activity are not likely responsible for the characteristic leaky nature of the tumor vascular barrier. The combined results from these studies therefore offer new insight into the characteristics of the vascular barriers in normal and tumor brain tissue and further elucidate the novel permeability effects of RMP-7. Together, they support its potential use as an adjunctive therapy for the selective delivery of chemotherapeutic drugs to brain tumors and possibly other neurodegenerative conditions.

Intravenous RMP-7 selectively increases uptake of carboplatin into rat brain tumors.

Rats implanted with RG-2 gliomas were administered i.v. RMP-7 and [14C]carboplatin. Changes in the permeability of the blood-brain barrier to carboplatin were determined using quantitative autoradiography. i.v. infusions of RMP-7 induced an increase in the permeability of the vascular barrier within the tumor to carboplatin. Additionally, permeability of brain tissue proximal to, but clearly outside the tumor mass, was also increased. Progressively less uptake of [14C]carboplatin was observed as distance from the tumor border increased. The increases in permeability induced by RMP-7 occurred in a dose-related fashion. No increase in carboplatin level was observed in several nonbrain tissues, including sciatic nerve, retina, heart, lung, liver, kidney, and spleen. Finally, the permeabilizing effects of RMP-7 were shown to occur independent of histaminergic or hypotensive mechanisms. These data provide additional insight into the permeabilizing effects and mechanism of RMP-7 and offer additional support for the therapeutic utility of this novel compound as an adjunctive treatment for human gliomas.

Lack of evidence for neutrophil participation during infarct formation following focal cerebral ischemia in the rat.

The involvement of neutrophils in the pathogenesis of cerebral ischemic injury in two rat models of focal ischemia was investigated. In Experiment I, a model of focal ischemia with partial reperfusion was used. Although significant and discrete ischemic damage within the neocortex was nearly maximal at 12 h postocclusion, no elevation in neutrophils was seen at this time point. Even after 21 h postocclusion, only a subtle increase in neutrophils within the ischemic tissue was observed. To further investigate the possible role of neutrophils in cerebral ischemia, the effect of cyclophosphamide-induced neutropenia was investigated (Experiment II). While a marked reduction (>98%) in systemic neutrophils was achieved in advance of and during the ischemic challenge, no reduction in the volume of ischemic damage was observed. In Experiment III, variations in the rat model of focal ischemia were made to produce a larger area of ischemic damage, as well as to permit complete reperfusion of blood to the affected cortex. While more neutrophils were seen in this variation of the model, very few were observed (< 1 per field) prior to the time that maximal ischemic damage had already occurred. Together, these experiments revealed that substantial brain necrosis occurred prior to the appearance of neutrophils, under conditions of partial, as well as complete, reperfusion. Moreover, at the time points when elevations in neutrophils were observed, no further increase in volume of ischemic damage was noted. Finally, pharmacologic removal of neutrophils prior to ischemia did not alter the size of the ischemic region. These data therefore fail to support the hypothesis that neutrophils play a general and essential role in infarct formation following focal brain ischemia and argue that further studies are required to more clearly elucidate the conditions under which neutrophils might participate in ischemic pathogenesis.

Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat.

Marked increases in intracellular calcium may play a role in mediating cellular dysfunction and death following central nervous system trauma, in part through the activation of the calcium-dependent neutral protease calpain. In this study, we evaluated the effect of the calpain inhibitor AK295 [Z-Leu-aminobutyric acid-CONH(CH2)3-morpholine] on cognitive and motor deficits following lateral fluid percussion brain injury in rats. Before injury, male Sprague-Dawley rats (350-425 g) were trained to perform a beam-walking task and to learn a cognitive test using a Morris water maze paradigm. Animals were subjected to fluid percussion injury (2.2-2.4 atm; 1 atm = 101.3 kPa) and, beginning at 15 min postinjury, received a continuous intraarterial infusion of AK295 (120-140 mg/kg, n = 15) or vehicle (n= 16) for 48 hr. Sham (uninjured) animals received either drug (n = 5) or vehicle (n = 10). Animals were evaluated for neurobehavioral motor function at 48 hr and 7 days postinjury and were tested in the Morris water maze to evaluate memory retention at 7 days postinjury. At 48 hr, both vehicle- and AK295-treated injured animals showed significant neuromotor deficits (P< 0.005). At 7 days, injured animals that received vehicle continued to exhibit significant motor dysfunction (P< 0.01). However, brain-injured, AK295-treated animals showed markedly improved motor scores (P<0.02), which were not significantly different from sham (uninjured) animals. Vehicle-treated, injured animals demonstrated a profound cognitive deficit (P< 0.001), which was significantly attenuated by AK295 treatment (P< 0.05). To our knowledge, this study is the first to use a calpain inhibitor following brain trauma and suggests that calpain plays a role in the posttraumatic events underlying memory and neuromotor dysfunction.

Calpain as a novel target for treating acute neurodegenerative disorders.

Calpains are cytosolic, neutral proteases that normally exist in an inactive or quiescent state. They require higher than normal levels of calcium for activation which, once accomplished, lead to irreversible proteolysis of numerous cytoskeletal, membrane-associated and regulatory proteins. Because of these characteristics, calpain is gaining attention as a potentially important pathogenic variable in ischemic neuronal death. This manuscript explores this hypothesis by briefly reviewing current support for the role played by calpain in ischemic neurodegeneration, and then discussing a series of recently published studies which: 1. offer further evidence for the hypothesis, and 2. provide direct support for the idea that selective inhibition of calpain can greatly limit the neuronal damage that would normally occur following both global as well as focal brain ischemia. Thus, the data reviewed in this manuscript support the ideas that unregulated activation and proteolysis of intraneuronal calpain plays a significant role in the brain damage that occurs following an ischemic event and that delivering selective and membrane permeant calpain inhibitors to ischemic tissue may provide a powerfully effective therapeutic means of limiting neuronal damage.

Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration.

BACKGROUND AND PURPOSE: This research was performed to determine whether a selective inhibitor of the calcium-dependent protease, calpain, could reduce ischemia-associated brain damage when peripherally administered after a vascular occlusion. METHODS: A variation of the rat middle cerebral artery occlusion model was used. A range of doses of AK295 (a novel calpain inhibitor synthesized for this purpose) was continuously infused through the internal carotid artery, beginning 1.25 hours from the initiation of the occlusion. Rats were killed at 21 hours, and the infarct volume was quantified. RESULTS: Postocclusion (1.25-hour) infusion of the calpain inhibitor AK295 elicited a dose-dependent neuroprotective effect after focal ischemia. The highest dose tested (3 mg/kg per hour) afforded the maximum effect, illustrated by a 32% reduction in infarct volume 21 hours after the ischemia (vehicle, 81.7 +/- 4.7 mm3; AK295, 54.9 +/- 6.9 mm3; P < .007). CONCLUSIONS: These data provide the first evidence that a peripherally administered calpain inhibitor can protect against ischemic brain damage. They offer further support for an important role of calpain proteolysis in the brain degeneration associated with cerebral ischemic events and suggest that selective calpain inhibitors provide a rational, novel, and viable means of treating such neurodegenerative problems.

The effect of dizocilpine and enadoline on immediate early gene expression in the gerbil global ischaemia model.

Increases in mRNA levels for c-fos, c-jun, junB, hsp70 and NGFI-A were observed in the dentate gyrus of the hippocampus following 7 min ischaemia in the Mongolian gerbil. The response was rapid and transient (30 min to 4 hr) for NGFI-A, junB and c-fos mRNA. In contrast c-jun mRNA remained increased for several hours. Hsp70 increased in the dentate gyrus 1 hr after the insult, returned to control values at 4 hr and showed a secondary increase at 24 hr. At 24 hr increased hsp70 mRNA was observed in other regions of the CNS, i.e. CA1, CA2, CA3 and cortex. The non-competitive NMDA receptor antagonist, dizocilpine, attenuated the increases in IEG expression and was neuroprotective. In contrast the kappa opiate receptor agonist, enadoline, protected the CA1 neurones from degeneration but did not inhibit the increased levels of IEG mRNA. Increases in hsp70 mRNA were reduced to baseline by both enadoline and dizocilpine. These results suggest that inhibition of IEG expression is not a prerequisite for neuroprotection. However, hsp70 was predictive of neuronal protection and may be a useful assay in this and related models.

Neuroprotective effect of the kappa-agonist enadoline (CI-977) in rat models of focal cerebral ischaemia.

The neuroprotective efficacy of the kappa-opioid agonist enadoline (CI-977) was examined in two acute rat models of focal cerebral ischaemia [non-recovery (4 h) and recovery (24 h)]. In the non-recovery model, Sprague-Dawley rats were anaesthetized throughout the study period. Focal ischaemia was produced by the permanent occlusion of the left middle cerebral artery (MCA). The amount of early ischaemic damage was assessed in coronal sections at nine pre-determined stereotaxic planes. Enadoline at doses of 0.1, 0.3 and 1.0 mg/kg (n = 8), administered s.c. 30 min prior to ischaemia, produced dose-dependent amelioration of cortical damage. Importantly, enadoline had no significant effect on any of the physiological parameters monitored (blood pressure, blood gases, glucose, pH). In the recovery model the left MCA was permanently occluded under isoflurane anaesthesia. Animals were allowed to recover and were killed 24 h later. The amount of ischaemic brain damage and swelling was assessed histologically. In this model pretreatment with enadoline at either 0.1, 0.3, or 1.0 mg/kg s.c. was followed by continuous s.c. infusion at 0.017, 0.05 or 0.17 mg/kg/h respectively (n = 8-17). Enadoline produced dose-dependent reductions in the volumes of infarction and brain swelling; the greatest reductions were seen at 1.0 mg/kg plus 0.17 mg/kg/h in both infarction (reduced by 37.4% from controls) and swelling (reduced by 47.8%). Therefore the kappa-opioid agonist enadoline affords dose-dependent neuroprotection in both the non-recovery and recovery models of focal cerebral ischaemia in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain temperature and the neuroprotective action of enadoline and dizocilpine in the gerbil model of global ischaemia.

Mongolian gerbils were subjected to transient forebrain ischaemia by occluding both common carotid arteries for 7 min. Subcutaneous administration of either the kappa-opioid receptor agonist enadoline (CI-977; 1 mg kg-1), or the non-competitive NMDA receptor antagonist dizocilpine (MK-801; 3 mg kg-1), at induction of ischaemia prevented neurodegeneration of CA1-CA2 pyramidal neurones in the dorsal hippocampus. It was shown by continuously monitoring intrahippocampal temperature that brain temperature drops by approximately 4 degrees C during ischaemia, when rectal temperature is maintained normothermic. Enadoline at no time point tested affected brain temperature, whereas dizocilpine statistically lowered brain temperature following ischaemia. These findings suggest that enadoline affords neuroprotection in the absence of any hypothermic episode, whilst in the case of dizocilpine, the small transient hypothermia observed following ischaemia may act synergistically or additively with the drug to yield neuroprotection.

The effect of CCKB/gastrin antagonists on stimulated gastric acid secretion in the anaesthetized rat.

1. The urethane-anaesthetized, vagotomised rat preparation was used to investigate the effects of the histamine H2-antagonist ranitidine, the proton pump inhibitor omeprazole and the CCKB/gastrin antagonists CI-988, PD 136450 and L-365,260 on pentagastrin-, histamine- and bethanechol-induced gastric acid secretion. 2. The novel CCKB/gastrin antagonists CI-988 and PD 136450, and L-365,260 dose-dependently inhibited pentagastrin-induced secretion. The ED50 value for PD 136450 was 0.05 mumol kg-1, the same following intravenous or subcutaneous administration. 3. CI-988 and PD 136450 administered subcutaneously at dose levels highly effective for antagonism of pentagastrin responses had no effect on basal acid secretion. 4. Ranitidine inhibited pentagastrin-, bethanechol-, and histamine-induced acid secretion, whereas the CCKB/gastrin antagonists inhibited only the secretory response to pentagastrin. 5. The selective CCKA antagonist, devazepide, was inactive at up to 300 mumol kg-1 i.p. against the three stimulants of acid secretion. 6. CI-988 and PD 136450 will be useful research tools with which to investigate the role of CCKB/gastrin receptors in gastric acid secretion and the trophic activities of gastrin and cholecystokinin (CCK) on the gastrointestinal tract.

Neuroprotective actions of GR89696, a highly potent and selective kappa-opioid receptor agonist.

1. The effect of a novel, highly potent and selective kappa-opioid receptor agonist, GR89696, has been evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in the Mongolian gerbil and permanent, unilateral middle cerebral artery occlusion in the mouse. 2. In the Mongolian gerbil model, administration of GR89696 (3 to 30 micrograms kg-1, s.c.), immediately before and at 4 h after insult, produced a dose-dependent reduction in the hippocampal CA1 neuronal cell loss resulting from a 7-min bilateral carotid occlusion. Similar effects were obtained with two other kappa-agonists, GR86014 (1 mgkg-1, s.c.) and GR91272 (1 mgkg-1, s.c.). The neuroprotective effect of GR89696 was completely blocked by prior administration of the opioid receptor antagonist, naltrexone, at 10 mgkg-1, s.c. Repeated post-treatment with GR89696 (100 micrograms kg-1, s.c.) or GR44821 (10 mgkg-1, s.c.) was also effective in protecting completely the hippocampal CA1 neurones from ischaemia-induced neurodegeneration. 3. In the permanent, unilateral middle cerebral artery occlusion model in the mouse, repeated administration of GR89696 at 300 micrograms kg-1, s.c. produced a 50% reduction in cerebrocortical infarct volume. In these experiments GR89696 was dosed 5 min, 4, 8, 12, 16, 20 and 24 h after occlusion on the first day and then three times daily for the next three days. GR89696 (300 micrograms kg-1) also produced a significant 35% reduction in infarct volume in this model when the initiation of dosing was delayed for 6 h after the insult. 4. The results indicate that the potent kappa-opioid receptor agonist, GR89696, is neuroprotective in both global and focal cerebral ischaemia models and suggest that, with this class of compound, there may be a considerable time window for pharmacological intervention.

A series of novel, highly potent and selective agonists for the kappa-opioid receptor.

1. This paper describes the opioid receptor pharmacology and in vivo activity of several novel benzene-acetamidopiperidine and benzeneacetamidopiperazine analogues. 2. These compounds all showed potent, naloxone-reversible, full agonist activity in the field-stimulated rabbit vas deferens, indicating that they are kappa-opioid agonists; but showed very little activity in the rat or hamster vas deferens, indicating good selectivity with regard to mu- and delta-opioid receptors. 3. They were all potent antinociceptive agents, the most potent compound, GR 103545, having an ED50 value in the mouse abdominal constriction test of 0.25 micrograms kg-1 s.c. The compounds also produced sedation and diuresis, but had little effect on respiration rate or gastrointestinal motility. 4. It is concluded that the seven novel compounds described are all potent and selective agonists for the kappa-opioid receptor.

Effect of inoculum size on ampicillin and amoxycillin susceptibility determined by gas-liquid chromatography for members of the family Enterobacteriaceae.

Inoculum size had only a small effect on the results for ampicillin and amoxycillin susceptibility testing of Escherichia coli. In contrast, a difference in inoculum size from 10(6) to 10(8) CFU/ml profoundly affected the results for ampicillin and amoxycillin susceptibility testing of Proteus mirabilis, causing a change from susceptibility to complete resistance. These different effects of inoculum size were observed both when susceptibility was determined by the suppression of a characteristic metabolic product analyzed by head-space gas-liquid chromatography (HS-GLC) and when it was determined by MIC testing in broth. Inoculum size affected the results for ampicillin susceptibility of P. mirabilis determined concurrently with the rapid HS-GLC urine test, because 10(8) CFU/ml may occur in urine specimens. In the rapid test, significant numbers of Escherichia, Klebsiella, Citrobacter, Proteus, Morganella, and Providencia spp. in urine specimens are detected within 4 h by HS-GLC analysis for characteristic metabolic products in cultures. Most P. mirabilis in urine specimens appeared to be ampicillin resistant in the rapid HS-GLC test but were reported to be ampicillin susceptible in hospital laboratory agar dilution tests 2 days later. However, ampicillin susceptibility results for Escherichia, Klebsiella, and Citrobacter spp. agreed with hospital laboratory reports. It was concluded that reports of ampicillin susceptibility or resistance for the commonest cause of urinary tract infections, E. coli, within 4 h of receipt of the specimen would be clinically valuable and that a provisional report of ampicillin resistance for P. mirabilis would not lead to ineffective therapy.

Volatile products from acetylcholine as markers in the rapid urine test using head-space gas-liquid chromatography.

Head-space gas--liquid chromatographic analyses of cultures of all Proteus spp. showed that the production of trimethylamine from acetylcholine could be the marker for the detection in 3.5 h of significant numbers of P. mirabilis, P. vulgaris, P. rettgeri and P. inconstans A in the rapid test for Escherichia and Klebsiella spp. in urine specimens. Trimethylamine was not detected in cultures of five other urinary pathogens. Six of fifteen strains of K. aerogenes produced trimethylamine from acetylcholine but were distinguished from Proteus spp. by ethanol production from arabinose. Ethyl acetate was produced from acetylcholine by P. mirabilis, P. vulgaris, P. rettgeri, P. inconstans A, P. inconstans B, E. coli, K. aerogenes and Streptococcus faecalis.

Head-space gas-liquid chromatography for the rapid laboratory diagnosis of urinary tract infections caused by enterobacteria.

Urine specimens were analysed in parallel in a hospital laboratory by routine methods which were regarded as the standard of correct diagnosis and by the rapid test already developed in a research laboratory. Technical modifications made to the rapid test ensured that its results agreed with those from routine methods and increased its rapidity so that a result was possible 4 h after receipt of the specimen. When 382 urine specimens were analysed by the modified test which is described in detail, there were neither false negatives nor false positives for infections with Escherichia, Klebsiella, Citrobacter or Proteus species.