Pituitary adenylate cyclase-activating polypeptide is a potent broad-spectrum antimicrobial peptide: Structure-activity relationships.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring cationic peptide with potent immunosuppressant and cytoprotective activities. We now show that full length PACAP38 and to a lesser extent, the truncated form PACAP27, and the closely related vasoactive intestinal peptide (VIP) and secretin had antimicrobial activity against the Gram-negative bacteria Escherichia coli in the radial diffusion assay. PACAP38 was more potent than either the bovine neutrophil antimicrobial peptide indolicidin or the synthetic antimicrobial peptide ARVA against E. coli. PACAP38 also had activity against the Gram-positive bacteria Staphylococcus aureus in the same assay with comparable potency to indolicidin and ARVA. In the more stringent broth dilution assay, PACAP38 had moderate sterilizing activity against E. coli, and potent sterilizing activity against the Gram-negative bacteria Pseudomonas aeruginosa. PACAP27, VIP and secretin were much less active than PACAP38 in this assay. PACAP38 also had some activity against the Gram-positive bacteria Bacillus cereus in the broth dilution assay. Many exopeptidase-resistant analogs of PACAP38, including both receptor agonists and antagonists, had antimicrobial activities equal to, or better than PACAP38, in both assays. PACAP38 made the membranes of E. coli permeable to SYTOX Green, suggesting a classical membrane lytic mechanism. These data suggest that analogs of PACPAP38 with a wide range of useful biological activities can be made by judicious substitutions in the sequence.

Pituitary adenylate cyclase-activating polypeptide (PACAP) protects against mitoxantrone-induced cardiac injury in mice.

Mitoxantrone (MXT) is an androstenedione that is used to treat cancers and progressive forms of multiple sclerosis; however, its use is limited by its cardiotoxicity. Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin/growth hormone-releasing hormone/vasoactive intestinal peptide family and has many functions, including cytoprotection and immunosuppression. We tested the hypothesis that PACAP can protect against MXT-induced cardiotoxicity in mice. Female BALB/c mice were treated once weekly for 4 weeks with saline (n=14) or MXT (3mg/kg, i.p.; n=14). Half of the mice in each group received PACAP (10µg, i.p.) 1h before and 24 and 48h after MXT, while the remaining mice received injections of saline on the same schedule. Echocardiography was used to assess cardiac structure and function. In mice treated with MXT and saline, body weight was significantly reduced after the third dose of MXT. PACAP significantly attenuated the reduction in body weight; however, the weights did not return to control level. Compared to controls, MXT-treated mice had significantly increased left ventricular (LV) diameter and LV volume and decreased LV posterior wall thickness. Fractional shortening (FS) and ejection fraction (EF) were also significantly decreased. Treatment with PACAP prevented MXT-induced LV dilation and significantly attenuated the reductions in FS and EF, although FS and EF did not return to control level. PACAP38 did not prevent MXT-induced decreases in LV posterior wall thickness. MXT dose-dependently decreased the viability of cultured U937 (human leukemia) cells; PACAP did not protect cultured U937 cells from MXT-mediated cell death. In conclusion, PACAP can attenuate MXT-mediated LV dilation and dysfunction in mice.

A structure-function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure-function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1-4, 14-17, 20-22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6-78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.

Pituitary adenylate cyclase-activating polypeptide prevents contrast-induced nephropathy in a novel mouse model.

We determined whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevents contrast-induced nephropathy using human renal proximal tubule epithelial (HK-2) cells and homozygous endothelial nitric oxide synthase-deficient (eNOS(-/-)) mice as a novel in vivo model. Cultured HK-2 cells were pretreated with 10(-9)-10(-6) mol/L PACAP or vasoactive intestinal peptide (VIP) for 1 h, and then exposed to ionic (Urografin) or nonionic (iohexol) contrast media at 50 mg iodine/mL for 24 h. Male eNOS(-/-) mice received Urografin (1.85 g iodine/kg) intravenously after water deprivation for 24 h, and PACAP38 (10 µg) intraperitoneally 1 h before and 12 h after Urografin injection. Urografin and iohexol increased lactate dehydrogenase and kidney injury molecule 1 in the culture medium, induced apoptosis, and inhibited cell proliferation in HK-2 cell cultures. PACAP38 and VIP reduced these changes in a dose-dependent manner. PACAP38 was more potent than VIP. In eNOS(-/-) mice, Urografin raised serum creatinine and cystatin C levels, caused renal tubule damage, induced apoptosis, and promoted neutrophil influx. Urografin also increased kidney protein levels of proinflammatory cytokines, and kidney mRNA levels of proinflammatory cytokines, kidney injury biomarkers, and enzymes responsible for reactive oxygen and nitrogen species. PACAP38 significantly reduced these Urografin-induced changes in eNOS(-/-) mice. This study shows that both Urografin and iohexol are toxic to HK-2 cells, but Urografin is more toxic than iohexol. Urografin causes acute kidney injury in eNOS(-/-) mice. PACAP38 protects HK-2 cells and mouse kidneys from contrast media and is a potential therapeutic agent for contrast-induced nephropathy.

Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors.

We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45 min. PACAP38, 20 µg in 100 µl of saline, was administered i.p. at 24 and 48 h after IR, and mice were euthanized at 72h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81 ± 0.08 vs. 0.69 ± 0.17 mg/dl in controls, p=NS, vs. 1.8 ± 0.03 in saline-treated IR mice, p<0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48 h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.

Renoprotection with pituitary adenylate cyclase-activating polypeptide in cyclosporine A-induced nephrotoxicity.

BACKGROUND: Acute and long-term nephrotoxicity is the major dose-limiting factor for cyclosporine A (CsA). We evaluated the protective effects of pituitary adenylate cyclase-activating polypeptide (PACAP)38 on CsA-induced nephrotoxicity in human renal proximal tubule epithelial (human kidney-2) cells and in intact mice. METHODS: Confluent (human kidney-2 cells were exposed to CsA (25-50 µmol/L) in the presence or absence of PACAP38 or vasoactive intestinal peptide (10(-10) to 10(-6) M). For studies in vivo, male BALB/c mice (n = 5 in each group) were given a single intraperitoneal injection of CsA (5 mg/kg body weight). Treatment group received 20 µg of PACAP38 2 hours before exposure to CsA and additional doses daily for 10 days. RESULTS: Cyclosporine A caused oxidative injury, marked morphological alterations, apoptosis, and increased expression of transforming growth factor (TGF)-ß1 in cell cultures. Pituitary adenylate cyclase-activating polypeptide 38 at 10(-8) mol/L restored cell confluency, reduced TGF-ß1 secretion, and preserved cell integrity. In mice, CsA caused tubular injury characterized by loss of tubular epithelial cell brush border membranes, tubular collapse, cellular necrosis, interstitial fibrosis, increased production of TGF-ß1, and elevated serum creatinine (3.39 ± 0.21 vs 0.13 ± 0.02 mg/dL in controls, P < 0.01). Treatment with PACAP38 reduced TGF-ß1 and tumor necrosis factor-α production in kidney, prevented epithelial-mesenchymal transition of the renal cells, and reduced serum creatinine levels to 1.01 ± 0.18 mg/dL, P < 0.01 versus CsA group. CONCLUSIONS: Pituitary adenylate cyclase-activating polypeptide 38 ameliorated renal tubular injury, reduced oxidative injury, and inhibited the expression of TGF-ß1 in CsA-exposed murine kidneys. Pituitary adenylate cyclase-activating polypeptide could be a novel renoprotective and antifibrotic agent for CsA nephrotoxicity.

Pituitary adenylate cyclase-activating polypeptide prevents cisplatin-induced renal failure.

Cisplatin is widely used for cancer chemotherapy, but nephrotoxicity is a major dose-limiting side effect. Our recent studies in vitro have shown that pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorated cisplatin nephrotoxicity and that the renoprotection with PACAP38 was mediated by the PAC(1) receptor and through the p53-dependent and -independent suppression of apoptosis of human renal proximal tubular epithelial cells. In the present studies, PACAP38 prevented the rise in blood urea nitrogen and serum creatinine in mice treated with cisplatin. Cisplatin-exposed mice treated with PACAP38 had relatively well-preserved tubular integrity, even when the treatment started 24 h after cisplatin exposure. PACAP38 also reduced plasma and kidney levels of tumor necrosis factor-α and restored collagen IV levels. The damage to mouse kidney tubules caused by cisplatin involved p53 accumulation and was partially reversed by treatment with PACAP38. PACAP38 ameliorates cisplatin-induced acute kidney injury even when treatment started 24 h after the onset of injury and increases tubular regeneration, which further facilitates restoration of kidney function in addition to its anti-apoptotic effects.

The effect of PACAP38 on MyD88-mediated signal transduction in ischemia-/hypoxia-induced acute kidney injury.

BACKGROUND/AIMS: toll-like receptor 4 (TLR4) and its adaptor protein MyD88 play an important role in ischemia/reperfusion (I/R) injury in the kidney, and pituitary adenylate cyclase-activating polypeptide (PACAP) could ameliorate renal I/R injury. METHODS: primary cultures of proximal tubule epithelial cells (PTEC) were prepared from wild-type and MyD88(-/-) mice, and subjected to hypoxia in vitro. Acute kidney injury (AKI) was induced by I/R in vivo in wild-type mice only. RESULTS: hypoxia resulted in significant increases in cytokine production and apoptosis/necrosis in wild-type PTEC, but these responses were markedly blunted in MyD88(-/-) PTEC. Treatment with PACAP38 before or after hypoxia further suppressed the hypoxia-induced cytokine responses and apoptosis in both MyD88(+/+) and MyD88(-/-) PTEC cultures. PACAP38 significantly inhibited TLR4/MyD88/TRAF6 as well as TRIF and IRF3 expression in mouse kidney and PTEC, and inhibited the secretion and mRNA expression of cytokines in kidneys from mice after I/R, paralleling the cytokine responses in vitro. Moreover, treatment with PACAP38 protected mice from renal failure, histological damage, neutrophil influx and tubule cell apoptosis after I/R. CONCLUSION: our data reveal that the TLR4-mediated cytokine responses to hypoxia are primarily dependent on MyD88 signaling and highlight the pivotal role of MyD88-dependent mechanisms in the coordination of the innate immune responses to ischemic/hypoxic acute renal tubular injury. The renoprotective effect of PACAP in AKI involves both MyD88-dependent and -independent pathways.

Myeloma light chain-induced renal injury in mice.

We investigated the effects of human light chain (LC) protein-overload in mice kidney to gain further insights into the molecular mechanisms involved in the pathogenesis of myeloma kidney. Intact male C57BL/6, 10- to 12-week-old mice were given daily intraperitoneal (i.p.) injections of 1 ml of human κ-LCs (1.5 mg/ml, low dose), or (100 mg/ml, high dose) to uninephrectomized mice for 2 weeks. Intact, sham-operated or uninephrectomized control animals were given the same volume (1 ml/day) of saline, human serum albumin (10 mg/ml) or bovine serum albumin (100 mg/ml) i.p. for 2 weeks in place of LCs. The low-dose LC-treated mice had human LCs in their urine and a significant increase in monocyte chemoattractant protein-1 (MCP-1) mRNA in the kidneys. Uninephrectomized mice treated with high-dose κ-LCs showed tubule casts, and foci of intracytoplasmic rhomboid crystals within the proximal tubules, along with cytoskeletal disruptions and alterations in the brush-border membrane, and high concentrations of human κ-LC were present in their sera. High-dose LC treatment also led to increases in serum creatinine and tumor necrosis factor-α levels, and marked increases in interleukin-6 and MCP-1 expression as well as cellular apoptosis in the kidneys. These studies demonstrate that myeloma LC overload over a range of LC concentrations in mice causes significant functional and morphological kidney injury. The model should be helpful in investigating pathophysiologic mechanisms and exploring therapeutic interventions for myeloma kidney and other LC-associated renal disorders.

Pituitary adenylate cyclase-activating polypeptide ameliorates cisplatin-induced acute kidney injury.

Cisplatin nephrotoxicity involves DNA damage, proinflammatory responses and apoptosis/necrosis of renal proximal tubular epithelial cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to protect kidneys from ischemic injury and light chain-induced damage by modulating inflammation. Confluent monolayer of HK-2 human renal cells were exposed to 50 microM cisplatin in the presence or absence of either PACAP38 or p53 siRNA. Mice injected with cisplatin were also treated with PACAP38 daily for 3 days. The damage to HK-2 cells caused by cisplatin involved the activation of p53, caspase-7, and poly (ADP-ribose) polymerase-1 (PARP-1). PACAP38 prevented the decrease in the apurinic/apyrimidinic endonuclease-1 by suppressing p53 activation and blocked the cleavage of caspase-7 and PARP-1 in cisplatin-exposed cells. PACAP also markedly inhibited cisplatin-induced apoptotic tubule cell death. Exposure to cisplatin significantly suppressed the expression of fibronectin and collagens I and IV, and altered the integrin repertoire of human renal tubule cells, while PACAP partially reversed the reduction of fibronectin, collagen IV, and the integrin subunits in cells exposed to cisplatin. Experiments with PACAP receptor antagonists and siRNA silencing of p53 showed that the renoprotection with PACAP was mediated by the PAC(1) receptor and through both p53-dependent and -independent suppression of apoptosis. PACAP was renoprotective in vivo and prevented the rise in blood urea nitrogen and creatinine in mice treated with cisplatin. These results suggest that p53 plays a pivotal role in decreased integrin-mediated extracellular matrix component expression in cisplatin-induced tubule cell apoptosis, and reveal a novel aspect of PACAP-mediated renoprotection.

Renoprotection by pituitary adenylate cyclase-activating polypeptide in multiple myeloma and other kidney diseases.

Renal involvement in patients with multiple myeloma complicates their treatment and shortens their life-span. The main renal lesion is a tubulointerstitial transformation with fibrosis, frequently associated with cast formation in the distal nephron that results from co-precipitation of pathological immunoglobulin light chains with Tamm-Horsfall proteins. The human renal proximal tubular reabsorption of excessive light chains by endocytosis causes cellular protein overload and activates the transcription factor nuclear factor kappa B (NFkappaB). The activation of NFkappaB promotes the synthesis of inflammatory cytokines and activates signaling pathways, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2, Jun kinase, and p38 MAPK, thus promoting interstitial inflammation and fibrosis. We tested the concept that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/vasoactive intestinal peptide family, could prevent the development of cast nephropathies. PACAP38 inhibited myeloma light chain-induced proinflammatory cytokine expression with greater potency than dexamethasone, and attenuated the resulting cell damage in the renal proximal tubule epithelial cells. The results indicated that its effects are mediated through inhibition of phosphorylation of p38 MAPK and nuclear translocation of the p50 subunit of NFkappaB via both the PAC(1) and VPAC(1) receptors. PACAP was also shown to be efficacious in other common in vivo animal models for kidney hypertrophies, including streptozotocin-induced diabetic nephropathy and gentamicin-induced nephrotoxicity. Thus, our studies suggest that PACAP38 could be used as a cytoprotective agent that would be effective in the treatment of renal tubule injury in multiple myeloma and other chronic kidney diseases.

Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study.

We have recently shown significant renoprotective effects with the administration of pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney. PACAP markedly inhibited the production of proinflammatory cytokines stimulated by immunoglobulin light chains in human renal proximal tubule epithelial cells and in the kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of 4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood rapidly declined after the cessation of administration with a half-life of about 5-10 min. The continuous infusion did not significantly alter the basal glucose level, blood gases or blood pressure. There was a large reduction in free lambda light chains in urine after the start of the treatment with PACAP. These studies show that PACAP can be safely used in humans and suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney.

Rapidly activated microglial cells in the preoptic area may play a role in the generation of hyperthermia following occlusion of the middle cerebral artery in the rat.

Postischemic hyperthermia occurs after the occlusion of the middle cerebral artery (MCAO) with an intraluminal filament in rats. The cause of hyperthermia is presumed to be damage to the preoptic area, which is one of the temperature-regulatory centers of the hypothalamus. In the present study, reactions of microglial cells and astrocytes in the preoptic area were examined during the first 6 h following transient MCAO. Microglial cells and astrocytes were visualized with immunohistochemistry using antibodies against the CR3 complement receptor and the glial fibrillary acidic protein, respectively. One hour after the occlusion, activated microglial cells were observed in both the medial and lateral preoptic areas ipsilaterally, and in the medial preoptic area contralateral to the infarct. Following reperfusion, the activation of microglial cells decreased in the medial preoptic area of both hemispheres, and in the lateral preoptic area there was a loss of immunoreactive microglial cells. Fragmentation of astrocytic processes was detected in the lateral preoptic area, while in the ipsilateral medial preoptic area a moderate swelling was observed. Immunohistochemistry with an antibody against interleukin-1beta (IL-1beta) revealed scattered immunoreactive cells in both the ipsilateral and the contralateral medial preoptic area 2 h after the MCAO. Our results show that microglial activation in the preoptic area coincides with postischemic hyperthermia. However, an exclusive role for IL-1beta in the generation of hyperthermia is unlikely, and other factors are probably also responsible for postischemic hyperthermia.

Distribution of urocortin-like immunoreactivity in the central nervous system of the frog Rana esculenta.

Corticotropin-releasing factor (CRF), sauvagine, and urotensin I are all members of the so-called CRF neuropeptide family. Urocortin (Ucn), a 40-amino-acid neuropeptide recently isolated from the rat brain, is the newest member of this family. Until now, the distribution of Ucn in the central nervous system (CNS) has been studied only in placental mammals. We used a polyclonal antiserum against rat Ucn to determine the distribution of Ucn-like immunoreactivity in the CNS of the green frog, Rana esculenta. The great majority of Ucn-immunoreactive perikarya was seen in the anterior preoptic area, ventromedial thalamic nucleus, posterior tuberculum, nucleus of the medial longitudinal fasciculus, and Edinger-Westphal nucleus. Urocortin-immunoreactive nerve cells were also observed in the motor nuclei of the trigeminal and facial nerves and in the hypoglossal nucleus. Immunoreactive fibers were found in the medial and lateral septal nuclei, bed nucleus of the stria terminalis, many of the thalamic and hypothalamic nuclei, mesencephalic tectum, tegmental nuclei, torus semicircularis, and dorsal horn and central field of the spinal cord. Only scattered Ucn-immunoreactive axon terminals were observed in the external zone of the medial eminence. The densest accumulations of Ucn-immunoreactive nerve terminals were seen in the granular layer of the cerebellum and cochlear nuclei. Our results suggest that an ortholog of mammalian Ucn occurs in the CNS of the green frog. The distribution of Ucn-like immunoreactivity in Rana esculenta showed many similarities to the distribution in placental mammals. The distribution of Ucn-like immunoreactivity in the anuran CNS was different from that of CRF and sauvagine, so our results suggest that at least three different lineages of the CRF neuropeptide family occur in the anuran CNS.

Distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of Rana esculenta.

The distribution of salmon and rat melanin-concentrating hormone (MCH)-like and neuropeptide glutamate-isoleucine (NEI)-like immunoreactivity in the brain and spinal cord of the frog Rana esculenta was studied with immunohistochemistry. In the telencephalon, only fibers showed immunoreactivity in the olfactory bulb, lateral pallium, diagonal band, septum, and the amygdala. Immunoreactive fibers were abundant in all diencephalic structures, except the optic tract, the visual neuropils, and the habenula. Several cells in the central thalamic nucleus and a few in the suprachiasmatic nucleus were stained with the MCH antisera. Cells and their processes were intensely stained in the dorsal hypothalamus with the MCH and NEI antisera. Immunoreactive fibers were found in all tegmental nuclei and the white matter of the mesencephalon. They formed terminal plexuses in the deep layers of the optic tectum and the laminar nucleus of the torus semicircularis. Immunoreactive fibers were sparse in the rhombencephalon and the spinal cord.

Filament size influences temperature changes and brain damage following middle cerebral artery occlusion in rats.

Postischemic spontaneous hyperthermia as a complication of occlusion of the middle cerebral artery with an intraluminal filament has been observed by some authors, but many other reports do not discuss this factor. The possible reasons why some of the authors have not seen severe hyperthermia in their experiments include differences in surgical technique, the strain of animals, the type of the anesthesia, and the occluder filament. The aim of this study was to examine the changes in the core temperature of rats using different types of filaments. The middle cerebral artery was occluded for 2 h with three different types of filaments. The changes in the temperature were continuously monitored during occlusion and for the next 4 h. Groups with uncontrolled hyperthermia and with controlled normal core temperature were used. In addition, the necrotic and penumbral areas were measured 4 and 48 h after the ischemia in both groups. Spontaneous postischemic hyperthermia was detected using all types of filaments. A close correlation was found between the size of the occluder filament and the time-course and degree of hyperthermia. Moreover, the size of the filament correlated well with the size of the infarct at both 4 and 48 h after the occlusion. We suggest that filament size is a major contributor to the degree of hyperthermia and the development of brain damage in the middle cerebral artery occlusion model. Our results call attention to the need to standardize the methods used to screen for therapeutic agents for stroke.