J. Kyle Krady

Diabetic Retinopathy Seeing Beyond Glucose-Induced Microvascular Disease

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Interleukin-1: A master regulator of neuroinflammation

Interleukins 1α and 1β (IL‐1) are very potent signaling molecules that are expressed normally at low levels, but are induced rapidly in response to local or peripheral insults. IL‐1 coordinates systemic host defense responses to pathogens and to injury and not surprisingly it has similar effects within the central nervous system (CNS). Numerous reports have correlated the presence of IL‐1 in the injured or diseased brain, and its effects on neurons and nonneuronal cells in the CNS, but it is only recently that the importance of IL‐1 signaling has been recognized. This article reviews studies that demonstrate that IL‐1 is at or near the top of the hierarchical cytokine signaling cascade in the CNS that results in the activation of endogenous microglia and vascular endothelial cells to recruit peripheral leukocytes (i.e., neuroinflammation). The IL‐1 system thus provides an attractive target for therapeutic intervention to ameliorate the destructive consequences of neuroinflammation.

The Type 1 Interleukin-1 Receptor Is Essential for the Efficient Activation of Microglia and the Induction of Multiple Proinflammatory Mediators in Response to Brain Injury

Interleukin-1 (IL-1) is induced immediately after insults to the brain, and elevated levels of IL-1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimers disease, and multiple sclerosis. In animal models, antagonizing IL-1 has been shown to reduce cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 IL-1 receptor (IL-1R1) to determine which cellular and molecular mediators of tissue damage require IL-1 signaling. At the cellular level, fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL-1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase-2 (Cox-2) and IL-6 expression were depressed and delayed. Interestingly, basal levels of Cox-2, IL-1, and IL-6 were significantly lower in the IL-1R1 null mice. In addition, stimulation of vascular cell adhesion molecule-1 mRNA was depressed in the IL-1R1 null mice, and correspondingly, there was reduced diapedesis of peripheral macrophages in the IL-1R1 null brain after injury. This observation correlated with a reduced number of Cox-2+ amoeboid phagocytes adjacent to the injury. In contrast, several molecular aspects of the injury response were normal, including expression of tumor necrosis factor-α and the production of nerve growth factor. Because antagonizing IL-1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self-propagating cycle of inflammation.

IGF‐I and microglia/macrophage proliferation in the ischemic mouse brain

We have used a model of hypoxic‐ischemic brain injury in adult male C57BL/6 mice to study insulin‐like growth factor‐I (IGF‐I) and IGF‐binding protein (IGFBP) expression in response to cerebral hypoxia‐ischemia (H/I) in the adult mouse. A period of 20 min of H/I that resulted in histopathology in cortex, striatum, and thalamus was correlated with induction of mRNA for IGF‐I, IGFBP‐2, IGFBP‐3, IGFBP‐5, and glial fibrillary acidic protein (GFAP) by 4 days of recovery. Increased IGF‐I mRNA was located within damaged regions and was surrounded by IGFBP‐2 mRNA expression. The results of combined immunostaining/in situ hybridzation showed that the cells expressing IGFBP‐2 mRNA were also GFAP‐positive and comprised a subset of activated astrocytes immediately surrounding areas of damage. In contrast, staining within damaged regions showed high numbers of cells immunopositive for F4/80 and lectin B4 indicative of microglia and macrophages but no cells immunopositive for the astrocytic proteins GFAP or S‐100β. Microglia/macrophages within the damaged areas expressed IGF‐I mRNA and were also immunopositive for the proliferating cell nuclear antigen. To determine whether expression of IGF‐I could contribute to proliferation of microglia, we treated purified cultures of adult brain microglia with IGF‐I in the presence of 3H‐thymidine. IGF‐I stimulated a twofold increase in DNA synthesis in cultures of adult brain microglia. Taken together with previous data demonstrating that IGF‐I promotes proliferation of peripheral macrophages, these data support the hypothesis that IGF‐I is an autocrine/paracrine mitogen for microglia/macrophages after H/I. GLIA 38:85–97, 2002.

Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury

Excessive inflammation has been implicated in the progressive neurodegeneration that occurs in multiple neurological diseases, including cerebral ischemia, and elevated levels of the proinflammatory cytokine interleukin-1 (IL-1) have been shown to exacerbate brain damage, whereas diminishing IL-1 levels limits the extent of injury. However, to date there is no consensus regarding which receptor(s) mediates the detrimental effects of IL-1. Because we have previously demonstrated that signaling through the IL-1 type 1 receptor (IL-1R1) is necessary for microglial activation and because results from other studies have implicated microglia as effectors of neurodegeneration, we hypothesized that inactivating the IL-1R1 would decrease the extent of damage caused by a hypoxic-ischemic (H/I) insult. It is shown that a mild insult initiates progressive neurodegeneration that leads to cystic infarcts, which can be prevented by inactivating the IL-1R1. The IL-1R1 null mice also show preserved sensorimotor function at 1 months recovery. The mild insult induces multiple proinflammatory cytokines and activates microglia, and these responses are dramatically curtailed in mice lacking the IL-1R1. Importantly, the neuroinflammation precedes the progressive enlargement of the infarct, suggesting that the inflammation is causal rather than a consequence of the brain damage. These findings show that abrogating the inflammation consequent to a mild H/I insult will prevent brain damage and preserve neurological function. Additionally, these data incriminate the IL-1R1 as a master proinflammatory cytokine receptor.

Transforming growth factor β1 prevents IL‐1β–induced microglial activation, whereas TNFα‐ and IL‐6–stimulated activation are not antagonized

Microglia rapidly respond to CNS injury, yet the mechanisms leading to their activation and inactivation remain poorly defined. In particular, few studies have established how interactions between inflammatory mediators affect the innate immune response of microglia. To begin to establish how microglia integrate signals from multiple inflammatory mediators, we examined the effects of interleukin 1β (IL‐1β), interleukin 6 (IL‐6), tumor necrosis factor α (TNFα), interferon γ (IFN‐γ), and transforming growth factor β1 (TGFβ1) on both newborn and bulk‐isolated adult microglia. To assess the functional state of the cells, we assayed the expression of cyclooxygenase 2 (Cox‐2), interleukin 6, and tumor necrosis factor α, and two protein tyrosine kinases that have been implicated in microglial responses to activational stimuli, HCK and FAK. These studies demonstrated that IL‐1β, TNFα, IL‐6, but not IFN‐γ increase the expression of Cox‐2, whereas they all increase the expression of HCK and FAK. In these studies, TGFβ1 either had no effect, or it decreased basal levels of these proteins. TGFβ1 blocked activation by IL‐1β when given prior to, or simultaneously with, IL‐1β. TGFβ1 blocked the induction of the tyrosine kinases, Cox‐2, and the induction of IL‐6 and TNFα mRNAs. However, TGFβ1 was ineffective in antagonizing the induction of Cox‐2 by either IL‐6 or TNFα. We conclude that the TGFβ receptor signaling cascades intersect with IL‐1, but they may not interact with IL‐6 or TNFα signaling pathways that lead to activation. GLIA 40:109–120, 2002.

Neuroinflammation and Both Cytotoxic and Vasogenic Edema Are Reduced in Interleukin-1 Type 1 Receptor-Deficient Mice Conferring Neuroprotection

Background and Purpose— Interleukin-1 (IL-1) is a proinflammatory cytokine implicated in multiple neurodegenerative diseases, including stroke. However, to date, there is no consensus regarding which receptor(s) mediates the detrimental effects of IL-1. We hypothesized that abrogating IL-1 type 1 receptor (IL-1R1) signaling would reduce edema, chemokine expression, and leukocyte infiltration; lower levels of iNOS; and, consequently, decrease free radical damage after mild hypoxia/ischemia (H/I), thus preserving brain cells. Methods— IL-1R1 null mice and wild-type mice were subjected to a mild H/I insult. MRI was used to measure the area affected at 30 minutes and 48 hours after H/I. An RNAse protection assay was used to evaluate changes in chemokine mRNA expression. RT-PCR was used to assess inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase mRNA levels. Immunohistochemistry was used to assess leukocyte infiltration. Western blots were used to assess iNOS and glutamate aspartate transporter protein levels. Results— IL-1R1 null mice had reduced cytotoxic and vasogenic edema. The volume of hyperintense signal on T2-weighted images was reduced on average by 90% at 48 hours after H/I. The induction of multiple chemokine mRNAs was significantly reduced in IL-1R1 null mice compared with wild-type mice at 18 and 72 hours after H/I, which correlated with fewer infiltrating CD3+ leukocytes. Levels of iNOS protein and mRNA (but not glutamate aspartate transporter) were significantly reduced in the IL-1R1 mouse brain. Conclusions— These findings indicate that abrogating IL-1R1 signaling could protect brain cells subsequent to a mild stroke by reducing edema and immune cell recruitment, as well as by limiting iNOS-mediated free radical damage.

Impaired Wound Healing after Cerebral Hypoxia—Ischemia in the Diabetic Mouse

Impaired peripheral wound healing is a hallmark of diabetis pathology and has been attributed to compromised macrophage activation. Stroke is another component of diabetic pathology, with increased tissue infarction and worsened recovery although the mechanisms remain unresolved. In this study, we investigated whether a compromised glial/macrophage response might contribute to cerebral hypoxic-ischemic (H/I) brain damage in diabetic (db/db), relative to their normoglycemic db/+ mice. Hypoxia-ischemia was induced in 8-week-old male db/db and db/+ mice by the ligation of right common carotid artery followed by systemic hypoxia (8% O2: 92% N2) for 17 mins. Mice were killed at specific intervals of reperfusion/recovery and the brains analyzed by in situ hybridization or total RNA isolation. In situ hybridization using bfl-1 (microglia) and glial fibrillary acidic protein (GFAP) (astrocytes) revealed expression of both bfl-1 and GFAP in the ipsilateral hemisphere at 4 h in the db/+ mice, which was delayed and minimal in the db/db mice. RNase protection assays showed a robust increase in expression of the proinflamatory cytokines tumor necrosis factor-α (TNFα), interleukin-1 IL-1α, and IL-1β mRNA in the db/+ mice at 6 to 8 h of reperfusion peaking at 8 to 12 h; in db/db mice expression was markedly delayed and diminished. Real-time-polymerase chain reaction (RT-PCR) confirmed the reduced and delayed expression TNFα, IL-1α, IL-1β, and the growth factors insulin-like growth factor-1 and ciliary neurotrophic factor in the db/db mice; enzyme-linked immunosorbent assays confirmed the reduced and delayed translation of IL-1β protein. These findings suggest that a compromised inflammatory response may underlie the greater infarct associated with diabetic db/db mice compared with their nondiabetic littermates following a hypoxic/ischemic insult.

Astrocytic ceruloplasmin expression, which is induced by IL-1β and by traumatic brain injury, increases in the absence of the IL-1 type 1 receptor

IL‐1α and IL‐1β are induced immediately after insults to the brain, and signaling through the type 1 IL‐1 receptor is essential for a normal microglial and astroglial response to injury. To better understand which genes are induced in astrocytes by IL‐1β, we used the unbiased technique of differential display to analyze mouse astroglial gene expression after IL‐1β treatment. Two novel genes were induced, as well as the gene for ceruloplasmin, a ferroxidase with antioxidant properties. Ceruloplasmin was analyzed further by Northern and Western blot. RNA and protein levels of ceruloplasmin were increased when astrocytes were treated with IL‐1β. To determine whether the IL‐1 type 1 receptor (IL‐1R1) is essential for the injury‐induced expression of ceruloplasmin, a Western blot analysis was performed after a traumatic brain injury on mice that were IL‐1R1‐deficient. Ceruloplasmin increased significantly above controls after injury; however, injury‐induced levels of ceruloplasmin were lower in IL‐1R1‐deficient (2.7‐fold increase) than in the wild‐type animals (3.5‐fold increase). These data indicate that while IL‐1R1 deletion has a slight effect on ceruloplasmin expression, it is not essential for either the basal or the induced expression of ceruloplasmin in vivo. Since ceruloplasmin buffers free copper, oxidizes ferrous iron, and catalyzes the dismutation of free radicals, increased levels of ceruloplasmin likely protect neurons and glia from sustaining damage after injury. Furthermore, as the IL‐1R1 has been proposed to be a target for achieving neuroprotection after injury, these data suggest that the protection afforded by ceruloplasmin will be retained even when the IL‐1R1 is antagonized.

Astrogliosis is delayed in type 1 interleukin-1 receptor-null mice following a penetrating brain injury

The cytokines IL-1α and IL-1β are induced rapidly after insults to the CNS, and their subsequent signaling through the type 1 IL-1 receptor (IL-1R1) has been regarded as essential for a normal astroglial and microglial/macrophage response. To determine whether abrogating signaling through the IL-1R1 will alter the cardinal astrocytic responses to injury, we analyzed molecules characteristic of activated astrocytes in response to a penetrating stab wound in wild type mice and mice with a targeted deletion of IL-1R1. Here we show that after a stab wound injury, glial fibrillary acidic protein (GFAP) induction on a per cell basis is delayed in the IL-1R1-null mice compared to wild type counterparts. However, the induction of chondroitin sulfate proteoglycans, tenascin, S-100B as well as glutamate transporter proteins, GLAST and GLT-1, and glutamine synthetase are independent of IL-1RI signaling. Cumulatively, our studies on gliosis in the IL-1R1-null mice indicate that abrogating IL-1R1 signaling delays some responses of astroglial activation; however, many of the important neuroprotective adaptations of astrocytes to brain trauma are preserved. These data recommend the continued development of therapeutics to abrogate IL-1R1 signaling to treat traumatic brain injuries. However, astroglial scar related proteins were induced irrespective of blocking IL-1R1 signaling and thus, other therapeutic strategies will be required to inhibit glial scarring.