Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

doi:10.1016/S0006-8993(00)03209-1

Brain Research

Volume 894, Issue 1, 9 March 2001, Pages 46-55

https://doi.org/10.1016/S0006-8993(00)03209-1 Get rights and content

Abstract

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5–2 μg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 μg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.

Introduction

Amelioration of neuronal cell damage is very important for prevention and cure of neurological disorders in cerebrovascular and neurodegenerative diseases. Occlusion of the bilateral common carotid artery of Mongolian gerbils for 5 min causes transient forebrain ischemia, which results in delayed neuronal death in the hippocampal CA1 region on the 4th day after the treatment [9]. This ischemic model has often been employed to determine the neurotrophic effect of chemicals because of the simple procedure required producing neuronal death. Recent studies have shown that administration of neurotrophic factors inhibits postischemic neuronal death [1], [11], [21], [24], [29]. Notably, intraventricular administration of nerve growth factor (NGF) [24] and basic fibroblast growth factor (bFGF) [21] ameliorate the delayed neuronal death in the CA1 hippocampal region following transient forebrain ischemia in Mongolian gerbils.

The present study was performed to evaluate whether midkine (MK) had neuroprotective or neurodegenerative activity in vivo on hippocampal delayed neuronal death in gerbils. MK is a heparin binding growth factor with a molecular weight of 13 kDa, first isolated as the product of a retinoic acid-responsive gene in an embryonal carcinoma cell differentiation system, and is rich in basic amino acids and cysteine [6], [7], [28]. Structurally, MK shares about 50% sequence identity with pleiotrophin (PTN, also called heparin binding growth associated molecule, HB-GAM) [12], [14], but is not related to other growth factors or neurotrophic factors.

Intense MK expression is observed in many tissues during midgestation periods in the mouse and rat [8], [16], [26], while its expression in adult tissues is generally weak. MK has various biological activities; it promotes neurite outgrowth [19], fibrinolytic activity of endothelial cells [10], survival of embryonic neurons [15] and migration of inflammatory leukocytes [4], [27] and embryonic neurons [13]. Gene knockout mice deficient in MK showed retarded postnatal development in the hippocampus [20]. Following experimental cerebral infarction in rats, MK expression is intensely induced in the surrounding edematic region, but not in the necrotic region, shortly after infarction [32], [34]. Its expression is also increased in the hippocampal CA1 subfield following transient forebrain ischemia in rats [17]. MK is known to prevent retinal degeneration induced by exposure to constant light [31]. These observations suggested that MK played important roles not only in neural development but also in the repair of neural tissue.

Recently, it was reported that NGF delays rather than prevents delayed neuronal death in the hippocampus after ischemia [5]. Therefore, we also tested whether MK delivered after ischemic insult was effective in preventing delayed neuronal cell death.

Section snippets

Midkine purification

To generate human MK protein, a cDNA fragment covering the open reading frame of human MK (nucleotide number 1-432, Ref. [30]) was inserted into the yeast expression vector pPIC9 (In Vitrogen, Leek, The Netherlands), followed by transfection of the recombinant plasmid into yeast (Pichia pastoris GS115; Research Corporation Technologies, Tucson, AZ) and selection with histidine and G418. Human MK protein secreted by the yeast into culture medium was purified to homogeneity by five successive

MK administration immediately before the bilateral common carotid arterial occlusion

MK solution was injected into the brain ventricle immediately before occlusion and HE-stained sections of the CA1 region were examined 7 days after treatment. The numbers of hippocampal CA1 neurons in the occluded and vehicle (physiological saline)-administered groups (Fig. 1a) were markedly reduced compared with those in the sham-operated group, which was used as a normal control (Fig. 1b). In the groups with occlusion and administration with 0.5–2 μg of MK in physiological saline (Fig. 1c–e),

Discussion

We used 20–26-week-old Mongolian gerbils, because large gerbils are necessary for blood collection.

The number of gerbils in this experiment was varied because of technical failures due to the gerbil’s death. The causes of death were as follows; (1) bleeding from carotid artery on removing clips for reperfusion, (2) respiratory arrest during forebrain ischemia processing, (3) dislocation of trachea on exposure of the carotid artery, and (4) rectal perforation during temperature determination. MK

Acknowledgements

This work was supported in part by Grants from the Ministry of Education, Science, Sports and Culture, Japan.

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Administration of MK protected against the early cell death caused by ischemia and attenuated cerebral infraction [30]. MK pretreatment reduced ischemia-induced neuronal cell death in the hippocampal CA1 region of gerbils [31]. Additionally, early post-ischemic training reduced the infract volume and neurological deficits and was associated with an increase in MK expression [2].
Preconditioning exercise can exert neuroprotective effects after stroke. However, the mechanism underlying these neuroprotective effects by preconditioning exercise remains unclear. We investigated the neuroprotective effects of preconditioning exercise on brain damage and the expression levels of the midkine (MK) and brain-derived neurotrophic factor (BDNF) after brain ischemia. Animals were assigned to one of 4 groups: exercise and ischemia (Ex), no exercise and ischemia (No-Ex), exercise and no ischemia (Ex-only), and no exercise and intact (Control). Rats ran on a treadmill for 30 min once a day at a speed of 25 m/min for 5 days a week for 3 weeks. After the exercise program, stroke was induced by a 60 min left middle cerebral artery occlusion using an intraluminal filament. The infarct volume, motor function, neurological deficits, and the cellular expressions levels of MK, BDNF, GFAP, PECAM-1, caspase 3, and nitrotyrosine (NT) were evaluated 48 h after the induction of ischemia. The infarct volume, neurological deficits and motor function in the Ex group were significantly improved compared to that of the No-Ex group. The expression levels of MK, BDNF, GFAP, and PECAM-1 were enhanced in the Ex group compared to the expression levels in the No-Ex group after brain ischemia, while the expression levels of activated caspase 3 and NT were reduced in the area surrounding the necrotic lesion. Our findings suggest that preconditioning exercise reduced the infract volume and ameliorated motor function, enhanced expression levels of MK and BDNF, increased astrocyte proliferation, increased angiogenesis, and reduced neuronal apoptosis and oxidative stress.
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Inflammatory cell infiltration and fibrin deposition play important roles in the development of crescentic glomerulonephritis (GN). In particular, activation of coagulation is an indispensable factor in crescent formation. However, the mechanisms underlying the pathogenesis of crescent formation have not been completely elucidated. We identified the growth factor midkine (MK) as a novel key molecule in the progression of crescentic GN induced by anti–glomerular basement membrane antibody. Despite the lack of significant differences in autologous and heterologous reactions, MK-deficient (Mdk^−/−) mice unexpectedly showed a greater number of necrotizing glomerular injuries than wild-type (Mdk^+/+) mice. Likewise, more tubulointerstitial damage was observed in Mdk^−/− mice, and this damage positively correlated with glomerular injury. Plasminogen activator inhibitor (PAI)-1 was strongly induced in the injured glomerulus of Mdk^−/− mice, particularly in crescents and endothelial cells. This enhanced PAI-1 production was associated with an increase in inflammatory cell infiltration and matrix deposition in the glomerulus and the interstitium of Mdk^−/− mice. In line with these in vivo data, primary cultured endothelial cells derived from Mdk^−/− mice exhibited higher PAI-1 mRNA expression on fibrin challenge and less fibrinolysis than Mdk^+/+ mice. In contrast, the expression of plasminogen activators was not affected. Our combined data suggest that MK leads to a blockade of PAI-1, which is closely associated with the suppression of crescentic GN.
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In various target cells, MK promotes cell growth, survival, and migration and gene expression [6–9]. MK is also involved in reproduction and repair of damaged tissues, as well as the pathogenesis of many diseases [10–12]. In particular, MK serves an important function in tumorigenesis and is more highly expressed in various epithelial malignancies than in normal tissues [13–16].
Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma.
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Midkine is a heparin-binding growth factor having various biological activities including chemotaxis of inflammatory cells, angiogenesis and migration of neuronal cells. These biological activities are expected to have a great impact on the pathology of brain infarction in subacute phase. Therefore, we investigated the effect of post-ischemic gene transfer of midkine in the phase.
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2007, European Journal of Pharmacology
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Research reportIntraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

Abstract

Introduction

Section snippets

Midkine purification

MK administration immediately before the bilateral common carotid arterial occlusion

Discussion

Acknowledgements

Neuron

Brain Res.

Biochem. Biophys. Res. Commun.

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Exp. Neurol.

Dev. Biol.

Brain Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Dev. Brain Res.

Dev. Brain Res.

Brain-derived neurotrophic factor protects against ischemic cell damage in rat hippocampus

J. Cereb. Blood Flow Metab.

Identification of programmed cell death in situ via specific labeling of DNA fragmentation

J. Cell Biol.

Neointima formation in a restenosis model is suppressed in midkine-deficient mice

J. Clin. Invest.

Research report
Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils