Granulocyte Colony-stimulating Factor In Rats Research Articles

Neuroprotective Effects of Direct Intrathecal Administration of Granulocyte Colony-Stimulating Factor in Rats with Spinal Cord Injury.

To date, no reliable methods have proven effective for treating spinal cord injury (SCI). Even systemic administration of methylprednisolone (MP) remains controversial. We previously reported that intrathecal (i.t.) administration of granulocyte colony-stimulating factor (G-CSF) improves outcome after experimental spinal cord ischemic insults in rats. The present study aimed to examine the neuroprotective efficacy of i.t. G-CSF or MP in rats with SCI. Female rats were subjected to spinal cord contusion injury at T10 using NYU impactor. We i.t. administered G-CSF (10 μg) or MP (one bolus of 100 μg, followed by 18 μg/h infusion for 23 h) immediately after SCI. Both G-CSF and MP significantly improved the rats' motor function after SCI. Immunofluorescence staining revealed suppressed expression of transforming growth factor-beta 1 (TGF-β1), chondroitin sulfate proteoglycans (neurocan and phosphacan), OX-42 and tumor necrosis factor alpha after i.t. G-CSF, but not MP, in rats with SCI. In addition, G-CSF significantly decreased the expression of astrocytic TGF-β1 and glial fibrillary acidic protein around the injury site. Furthermore, rats with G-CSF treatment showed increased neurofilament expression beyond the glial scars. Direct i.t. administration of G-CSF provides a promising therapeutic option for SCI or related spinal diseases.

Abstract 353: Pharmacokinetic studies on a novel recombinant human granulocyte colony stimulating factor in rats

Abstract Background: Recombinant human granulocyte colony stimulating factor (G-CSF) has been used as a therapeutic drug for leukopenia of cancer patients who receive myelo-suppressive radio- or chemotherapy. Due to its intrinsic instability, G-CSF has to be administered to patients frequently to maintain high plasma concentrations for therapeutic effects. We report here that a recombinant G-CSF derivative (rhG-CSFa), which was previously found to exhibit a greater biological activity than wild-type G-CSF does, displays enhanced stability in vitro, and superior bioavailability in vivo. Methods: The pharmacokinetics of rhG-CSFa and G-CSF was investigated in rats; various groups of the rats received either intravenous (i.v.) or subcutaneous (s.c.) injection of rhG-CSFa, at one of three different doses (20, 50 or 100 µg/kg). The serum samples were collected at multiple time points after the drug administration (from 0.08 to 12 h). The concentrations of rhG-CSFa and wild-type G-CSF in various serum samples were determined by a sandwich ELISA. Pharmacokinetic parameters were analyzed by fitting the serum drug concentration-time data to a one- or two-compartment model, with use of the Winnonlin 5.2 software. For proteolytic studies in vitro, the concentrations of rhG-CSFa or G-CSF were determined at 3-min intervals after addition of the respective drug to rat serum or plasma. Results: Pharmacokinetic analysis of serum rhG-CSFa or G-CSF levels indicated that, at each dose tested, for either routes of drug administration, the area under concentration-time curve (AUC) values were higher for rhG-CSFa than for G-CSF. For example, at the dose of 20 µg/kg, s.c., the AUC value was 850 ± 80 ng*hr/ml for rhG-CSFa and 430 ± 50 for ng*hr/ml for G-CSF (n=5). At the dose of 20 µg/kg, i.v., the AUC value was 1150 ± 480 ng*hr/ml for rhG-CSFa and 810 ± 150 for ng*hr/ml for G-CSF (n=5). Differences in other pharmacokinetics parameters, including serum half-life (t1/2) and maximum serum concentration (Cmax), were also observed, further indicating greater serum bioavailability of the rhG-CSFa, as compared to G-CSF. Results from subsequent in vitro serum and plasma stability studies showed that the rates of drug disappearance were slower for rhG-CSFa than for G-CSF, when the respective drug was added to either serum (by 1.5 fold) or plasma (by 1.8 fold). This finding suggested that the rates of proteolysis of the rhG-CSFa were much slower, as compared to the rates of proteolysis of the wild-type G-CSF, either in serum or in plasma. Conclusions: The novel rhG-CSFa has a much better serum and plasma stability in vitro and a much higher bioavailability in vivo, as compared to the conventional G-CSF. The increased stability and bioavailability of the rhG-CSF may explain, at least partly, the greater therapeutic efficacy of rhG-CSFa than that of G-CSF in rats, and suggests that rhG-CSFa will also have enhanced therapeutic efficacy in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 353.

Synergetic Effects of Granulocyte-Colony Stimulating Factor and Cognitive Training on Spatial Learning and Survival of Newborn Hippocampal Neurons

Granulocyte-Colony Stimulating Factor (G-CSF) is an endogenous hematopoietic growth factor known for its role in the proliferation and differentiation of cells of the myeloic lineage. Only recently its significance in the CNS has been uncovered. G-CSF attenuates apoptosis and controls proliferation and differentiation of neural stem cells. G-CSF activates upstream kinases of the cAMP response element binding protein (CREB), which is thought to facilitate the survival of neuronal precursors and to recruit new neurons into the dentate gyrus. CREB is also essential for spatial long-term memory formation. To assess the role and the potential of this factor on learning and memory-formation we systemically administered G-CSF in rats engaged in spatial learning in an eight-arm radial maze. G-CSF significantly improved spatial learning and increased in combination with cognitive training the survival of newborn neurons in the hippocampus as measured by bromodeoxyuridine and doublecortin immunohistochemistry. Additionally, G-CSF improved re-acquisition of spatial information after 26 days. These findings support the hypothesis that G-CSF can enhance learning and memory formation. Due to its easy applicability and its history as a well-tolerated hematological drug, the use of G-CSF opens up new neurological treatment opportunities in conditions where learning and memory-formation deficits occur.

Mobilization of Mesenchymal Stem Cells by Granulocyte Colony-stimulating Factor in Rats with Acute Myocardial Infarction

Intravenous delivery of mesenchymal stem cells (MSCs), a noninvasive strategy for myocardial repair after acute myocardial infarction (MI), is limited by the low percentage of MSCs migration to the heart. The purpose of this study was to test whether granulocyte colony-stimulating factor (G-CSF) would enhance the colonization of intravenously infused MSCs in damaged heart in a rat model of acute MI. After induction of anterior MI, Sprague-Dawley rats were randomized to receive: (1) saline (n=9); (2) MSCs (n=15); and (3) MSCs plus G-CSF (50 microg/kg/day for 5 consecutive days, n=13). Flow cytometry revealed that G-CSF slightly increased surface CXCR4 expression on MSCs in vitro. After completion of G-CSF administration, MSCs showed a significantly lower colonization in bone marrow and a trend toward higher localization in the infarcted myocardium. At 3 months, vessel density in the infarct region of heart was significantly increased in MSCs group and trended to increase in MSCs+G-CSF group. However, echocardiographic and hemodynamic parameters, including left ventricular (LV) end-diastolic diameters, ejection fraction, and +/-dP/dtmax, were not statistically different. Morphological analysis showed that infarct size and collagen content were similar in the three groups. Immunohistochemistry revealed that the combined therapy accelerated endothelial recovery of the blood vessels in the ischemic myocardium. However, myocardial regeneration resulting from MSCs differentiation was not observed. G-CSF enhanced the migration of systemically delivered MSCs from bone marrow to infarcted heart. However, the beneficial effect of this kind of migration is limited, as cardiac function did not improve.

An extended window of opportunity for G-CSF treatment in cerebral ischemia.

BackgroundGranulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models.ResultsHere, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 μg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 μg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints.ConclusionThese data further strengthen G-CSF's profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase.

Development and validation of a sensitive ELISA for quantitation of Grastim® (rhG‐CSF) in rat plasma: application to a pharmacokinetic study

Grastim is bacterially produced recombinant counterpart of human granulocyte colony stimulating factor (G-CSF). It has biological activity similar to that of endogenous G-CSF. In the present work a sensitive, accurate, precise and enzyme-linked immunosorbent assay (ELISA) for the quantitation of G-CSF in rat plasma was developed and validated. The ELISA method employed a technique in which anti-human-G-CSF was adsorbed onto 96-well maxisorp plates and used to capture the G-CSF in rat plasma samples. The captured G-CSF was then detected using streptavidin-HRP amplification system. Absolute recovery was >90% from rat plasma. The validation includes assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLOQ), storage stability (30 days) in rat plasma and assay specificity. The standard curve for G-CSF was linear (R2 > 0.996) in the concentration range 4.88-625 pg/mL. The LLOQ was established at 4.88 pg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 15, 250 and 500 pg/mL, were in the range 3.00-8.66% relative standard deviation (RSD) and 1.03-4.69% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.28-110.79% of the nominal values. The assay shows dilutional linearity and specificity. Stability of G-CSF was established for 30 days at -80 degrees C and through three freeze-thaw cycles. The validated assay was successfully employed for the assessment of pharmacokinetic disposition of G-CSF in rats.

Splenomegaly induced by recombinant human granulocyte-colony stimulating factor in rats

Spontaneous ruptures of the spleen have been observed in donors and patients with malignancy during mobilization of peripheral blood stem cells. Thus, we investigated the morphological and histological alteration of the spleen, and mRNA expression levels and activities of the DNA-synthesizing enzymes thymidylate synthase (TS) and thymidine kinase (TK) in the splenic cells, of rats treated with recombinant human granulocyte-colony stimulating factor (rhG-CSF). Daily injections of rhG-CSF for 5 or 7 days slightly enhanced the splenic weight. Single or 3-day treatment with rhG-CSF markedly enhanced the number of granulocytes in peripheral blood. Expression levels of TS and TK mRNA in the splenic cells were significantly increased 6 hours after rhG-CSF treatment. Early expression of TS and TK mRNA in the splenic cells may indicate a reseeding of hematopoietic cells from the bone marrow. Daily injections with rhG-CSF enhanced the TS and TK activities in the splenic cells. As continuous elevations of DNA-synthesizing enzyme activity and spleno-megaly are suggestive of a possible splenic rupture, the monitoring of peripheral granulocytes and splenic size is necessary during the rhG-CSF treatment.

Pituitary–adrenocortical responses to the chronic administration of granulocyte colony-stimulating factor in rats

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor, but it may play a role in the regulation of the neuroendocrine system activity. Only few data are available about its possible influence on the pituitary gland. We have recently reported an acute stimulatory effect of G-CSF (and of GM-CSF) on adrenocorticotropic hormone (ACTH) secretion in rats in vivo. The purpose of the present study was to evaluate whether chronic administration of G-CSF affects ACTH and corticosterone secretion and growth processes of the rat anterior pituitary gland and adrenal cortex in vivo. We have demonstrated that G-CSF (at a dose of 10.0 μg/kg body weight (BW)) injected s.c. once daily (for 7 days), stimulated both ACTH and corticosterone secretion. Simultaneously, G-CSF treatment did not change the total anterior pituitary cell proliferation as revealed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). On the other hand, proliferative activity of corticotrophs, detected in the sections of the anterior pituitary using double-labeling, was significantly increased after treatment with G-CSF. Moreover, this growth factor induced an increase in the proliferation ratio in the entire adrenal equatorial section. These findings suggest an involvement of G-CSF in the regulation of pituitary–adrenal axis and support the hypothesis of bidirectional associations between the immune system and the endocrine glands.

Enhancement of peritoneal leukocyte function by granulocyte colony-stimulating factor in rats with abdominal sepsis.

To investigate the therapeutic effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and peritoneal neutrophils during intra-abdominal sepsis. Placebo, controlled study, using a rat model of intra-abdominal sepsis. Animal research facility. Male specific pathogen-free Sprague-Dawley rats. Abdominal sepsis was produced in rats by cecal ligation and puncture. The control animals received a sham operation. G-CSF (subcutaneous injection at 50 microg/kg) or vehicle (100 microL of 5% dextrose) treatment was initiated at 1 hr after cecal ligation and puncture or sham operation and repeated at 12-hr intervals thereafter. Six hours after cecal ligation and puncture, CD11b/c and CD18 expression on circulating neutrophils was significantly up-regulated when compared with those in the sham operated control animals. Peritoneal neutrophils exhibited a further up-regulation of these adhesion molecules than did the circulating neutrophils. A sustained up-regulation of CD11b/c and CD18 was found in peritoneal neutrophils even at 24 hrs after cecal ligation and puncture. G-CSF treatment increased CD11b/c expression on circulating neutrophils in 6-hr sham-operated rats, but did not further up-regulate CD11b/c or CD18 expression on circulating or peritoneal neutrophils in cecal ligation and puncture rats. Phagocytic activities of circulating neutrophils assessed by uptake of fluorescent latex microspheres were lower in 24-hr cecal ligation and puncture rats when compared with the sham-operated controls. G-CSF treatment prevented this inhibition. Furthermore, G-CSF enhanced the phagocytic activities of peritoneal neutrophils in both 6- and 24-hr cecal ligation and puncture rats when compared with those of the vehicle-treated animals. Spontaneous hydrogen peroxide generation by circulating neutrophils was increased in 6-hr cecal ligation and puncture rats, but not in 24-hr cecal ligation and puncture rats. Peritoneal neutrophils exhibited an inhibition of phorbol myristate acetate-stimulated hydrogen peroxide generation. G-CSF treatment did not up-regulate neutrophil hydrogen peroxide generation. Circulating and peritoneal neutrophils exhibit marked polymorphism in their functional activities during the host response to abdominal sepsis. G-CSF treatment significantly enhanced the phagocytic function of both circulating and peritoneal neutrophils which may be one mechanism underlying its protective effect in abdominal sepsis.

Histopathological study on bone changes induced by recombinant granulocyte colony-stimulating factor in rats

Histopathological bone changes were examined in growing rats intravenously administered with high doses (100 and 1000 micrograms/kg/day) of recombinant human granulocyte colony-stimulating factor (rG-CSF) for 28 days. The changes were observed in the region where physiological bone resorption actively occurs in the growth phase, that is the trabeculae of metaphyseal spongy bone and the endosteum region of diaphyseal compact bone. Histologically, the changes involved accelerated osteoclastic bone resorption and osteogenesis due to intramembranous ossification. While osteoclastic bone resorption was observed in almost all lesions, about half of which were accompanied by osteogenesis. Bone changes which appeared after administration of rG-CSF were characterized by frequent occurrence at the site of highly osteoclastic activity and by initial osteoclastic resorption followed by osteogenesis due to intramembranous ossification. These results suggest that the main action of rG-CSF on bone may be an acceleration of osteoclastic bone resorption.

Prolonged expression of therapeutic levels of human granulocyte colony-stimulating factor in rats following gene transfer to skeletal muscle.

Gene transfer to skeletal muscle was examined as a means of gene therapy for neutropenia. A recombinant retrovirus containing a human granulocyte colony-stimulating factor (G-CSF) gene was introduced into primary human or rat myoblasts, which were then shown to produce biologically active G-CSF. Transplantation of G-CSF-producing rat myoblasts into the muscle of syngeneic rats resulted in a 15-fold increase in absolute neutrophil counts. This increase correlated with detection of circulating human G-CSF protein throughout the 6-month duration of the experiment. These results clearly demonstrate long-term production of therapeutically relevant amounts of a human protein by normal cells in vivo.

Effects of a dosing solution on the nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor in rats.

The nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor produced by Esherichia coli (E. coli-rhG-CSF) was studied in rats. We investigated the effects of the basic conditions of the dosing solution and the enhancing effects of various substances to find means to improve the nasal absorption of E. coli-rhG-CSF. Relative bioavailability following the nasal administration of E.coli-rhG-CSF solution to subcutaneous administration was 3.6%. Osmotic pressure of the dosing solution had virtually no effect on the nasal absorption of E. coli-rhG-CSF. Absorption was enhanced by a decrease in pH, an increase in buffer concentration at pH2 and an increase in drug concentration. Sodium cholate, ascorbic acid, tartaric acid, beta-cyclodextrin and dimethyl-beta-cyclodextrin in remarkably increased the nasal absorption of E. coli-rhG-CSF. Condition adjustments to the dosing solution and the use of several enhancers were significantly concluded to increase the intranasal absorption of E. coli-rhG-CSF.