Interleukin-4-Mediated Oxidative Stress Is Harmful to Hippocampal Neurons of Prothrombin Kringle-2-Lesioned Rat In Vivo

Abstract

The present study investigated the effects of reactive microglia/macrophages-derived interleukin-4 (IL-4) on hippocampal neurons in prothrombin kringle-2 (pKr-2)-lesioned rats. pKr-2 was unilaterally injected into hippocampus in the absence or presence of IL-4 neutralizing antibody (IL-4Nab). Immunohistochemical analysis showed a significant loss of Nissl+ and NeuN+ cells and activation of microglia/macrophages (increase in reactive OX-42+ and OX-6+ cells) in the hippocampus at 7 days after pKr-2 injection. The levels of IL-4 expression were upregulated in the reactive OX-42+ microglia/macrophages as early as 1 day, maximal at 3 days and maintained up to 7 days after pKr-2 injection. Treatment with IL-4Nab significantly increased neuronal survival in pKr-2-treated CA1 layer of hippocampus in vivo. Accompanying neuroprotection, IL-4 neutralization inhibited activation of microglia/macrophages, reactive oxygen species-derived oxidative damages, production of myeloperoxidase- and inducible nitric oxide synthase-derived reactive nitrogen species and nitrosative damages as analyzed by immunohistochemistry and hydroethidine histochemistry. These results suggest that endogenous IL-4 expressed on reactive microglia/macrophages mediates oxidative/nitrosative stress and play a critical role on neurodegeneration of hippocampal CA1 layer in vivo.

Interleukin-13 Propagates Prothrombin Kringle-2-Induced Neurotoxicity in Hippocampi In Vivo via Oxidative Stress

“…When NeuN + cells on the ipsilateral side were quantified, it was found that IL-13Nab significantly increased the number of NeuN + cells in the CA1 layer of the hippocampus compared with pKr-2 only ( Figure 3 I). As controls, IL-13Nab alone ( Figure 3 I) and nonspecific goat IgG in the absence ( Figure 3 G,H) or presence of pKr-2 [ 16 ] (data not shown) did not affect neuronal survival [ 16 , 19 , 29 , 30 ], similar to that observed with PBS or pKr-2 only.…”

“…Activated microglia/macrophages and neutrophils produce inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO), which induces oxidative/nitrosative stress, resulting in neurodegeneration [ 4 , 16 ]. Thus, we examined whether intrahippocampal injection of pKr-2 induced expression of iNOS and MPO in activated microglia/macrophages and neutrophils.…”

“…For pKr-2 injection, rats (10-week-old. 240–260 g) were anesthetized by intraperitoneal injection of chloral hydrate (360 mg/kg; Sigma-Aldrich., St. Louis, MO, USA) and immobilized on stereotaxic instruments [ 16 , 17 ]. According to the atlas of Paxinos and Watson (ref), rats received unilateral injection of 48 μg pKr-2 (Haematologic Technologies., Essex Junction, VT, USA) in 4 μL of phosphate-buffered saline (PBS; Gibco., Palsey, UK) into the right CA1 of hippocampus (3.6 mm posterior to bregma, 2.0 mm lateral to the midline, 2.8 mm beneath the skull of the brain) with 30 gauge of Hamilton syringe at a rate of 0.2 μL per minute.…”

“…Levels of pKr-2 and thrombin were elevated in the postmortem brains of AD and PD patients [ 21 , 22 ], suggesting that pkr-2 might be involved in neuropathological processes as an endogenous neurotoxin. Regarding this, we also demonstrated that pKr-2-induced microglial activation and microglial proinflammatory cytokines cause neurodegeneration in rat cortices, substantiae nigrae, and hippocampi in vivo [ 16 , 17 , 18 ].…”

“…Prothrombin kringle-2 (pKr-2) is the second domain of prothrombin and is derived from prothrombin to active thrombin. pkr-2 is known to induce microglial activation, ROS/RNS generation, and production of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in vivo and in vitro [ 16 , 17 , 18 , 19 , 20 ]. Levels of pKr-2 and thrombin were elevated in the postmortem brains of AD and PD patients [ 21 , 22 ], suggesting that pkr-2 might be involved in neuropathological processes as an endogenous neurotoxin.…”