Rose A. Korsak

STAT3 is a Critical Regulator of Astrogliosis and Scar Formation after Spinal Cord Injury

Signaling mechanisms that regulate astrocyte reactivity and scar formation after spinal cord injury (SCI) are not well defined. We used the Cre recombinase (Cre)-loxP system under regulation of the mouse glial fibrillary acidic protein (GFAP) promoter to conditionally delete the cytokine and growth factor signal transducer, signal transducer and activator of transcription 3 (STAT3), from astrocytes. After SCI in GFAP-Cre reporter mice, >99% of spinal cord cells that exhibited Cre activity as detected by reporter protein expression were GFAP-expressing astrocytes. Conditional deletion (or knock-out) of STAT3 (STAT3-CKO) from astrocytes in GFAP-Cre-loxP mice was confirmed in vivo and in vitro. In uninjured adult STAT3-CKO mice, astrocytes appeared morphologically similar to those in STAT3+/+ mice except for a partially reduced expression of GFAP. In STAT3+/+ mice, phosphorylated STAT3 (pSTAT3) was not detectable in astrocytes in uninjured spinal cord, and pSTAT3 was markedly upregulated after SCI in astrocytes and other cell types near the injury. Mice with STAT3-CKO from astrocytes exhibited attenuated upregulation of GFAP, failure of astrocyte hypertrophy, and pronounced disruption of astroglial scar formation after SCI. These changes were associated with increased spread of inflammation, increased lesion volume and partially attenuated motor recovery over the first 28 d after SCI. These findings indicate that STAT3 signaling is a critical regulator of certain aspects of reactive astrogliosis and provide additional evidence that scar-forming astrocytes restrict the spread of inflammatory cells after SCI.

Fatty Acid Oxidation and Ketogenesis by Astrocytes in Primary Culture

Abstract: The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D‐(–)‐3‐hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2‐day‐old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3–0.5 mM) was 50–90 nmol C2 units h−1 mg of protein−1. A similar rate was obtained using radiolabeled tracer methodology with [1‐14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1‐14C]‐ and [7‐14C]octanoate and [1‐14C]‐, [13‐14C]‐, and [15‐14C]palmitate indicated that a substantial proportion of the ω‐terminal fourcarbon unit of these fatty acids bypassed the β‐ketothiolase step of the β‐oxidation pathway and the 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1‐14C‐labeled fatty acids, obligated to pass through the acetyl‐CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (ω‐1)‐labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (ω‐3)‐labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3‐oxo‐acid‐CoA transferase (EC 2.8.3.5) or acetoacetyl‐CoA deacylase (EC 3.1.2.11) or both on acetoacetyl‐CoA and not by the action of the mitochondrial HMG‐CoA cycle involving HMG‐CoA lyase (EC 4.1.3.4), which was readily detected, and HMG‐CoA synthase (EC 4.1.3.5), which was barely measurable.

Biocompatibility of amphiphilic diblock copolypeptide hydrogels in the central nervous system.

Amphiphilic diblock copolypeptide hydrogels (DCHs) are synthetic materials whose properties can be varied readily and predictably by altering copolymer chain length or composition and which are of potential interest for biomaterial applications. We tested the biocompatibility in the central nervous system (CNS) of DCH composed of lysine, homoarginine or glutamate in combination with leucine. A range of DCH formulations with rheological properties similar to brain tissue were injected into mouse forebrain and examined after 1-8 weeks using light microscopy, immunohistochemistry and electron microscopy. DCH deposits elicited no more gliosis, inflammation, or toxicity to neurons, myelin or axons than did injections of physiological saline. The size, rigidity, and density of DCH deposits could be varied subtly by altering DCH composition and concentration. For any given DCH formulation, increased concentration correlated with increased gel strength in vitro and increased deposit size in vivo. DCHs of lysine and leucine (K(m)L(n)) were selected for detailed analyses because these formed deposits with desirable physical properties and since lysine is routinely used as a substrate for neural cell cultures. Deposits of unmodified K(180)L(20) exhibited time-dependent in-growth of blood vessels and of certain glial cells, and limited in-growth of nerve fibers. These findings show that DCHs are injectable, re-assemble in vivo to form 3-dimensional deposits, exhibit little or no detectable toxicity in the CNS, integrate well with brain tissue and represent a new class of synthetic biomaterials with potential for applications as depots or scaffolds in the CNS.

Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat

1. Procedures are described to prepare nutritionally adequate rat milk-substitutes by modifying commercially available processed cows milk, rich in carbohydrate and low in protein and fat compared with rats milk. 2. Premilk formulas, prepared as intermediates in the preparation of rat milk-substitutes, are rich in protein but low in their concentration of fat, carbohydrate, and minerals when compared with rats milk. 3. Premilks were supplemented with lactose, vitamins, minerals, fat as oil mixtures, certain amino acids and other constituents to yield rat milk-substitutes which resemble the known composition of rats milk in their properties and composition. 4. Detailed analyses of the milk-substitutes show them to be comparable to rats milk in energy content, pH, osmolarity, the concentration of the macronutrients, fat, protein and carbohydrate, and the major minerals. 5. Rat pups were artificially reared from postnatal day 4 or 5 until days 16-18 by fitting them with gastric cannulas through which the milk-substitutes could be infused automatically. 6. The nutritional impact of the milk-substitutes was assessed by a comparison of growth and metabolic characteristics for artificially reared rats with age-matched sucking rats reared by their mother. 7. Indices which were taken to be appropriate included (a) body-weight gain; (b) the concentration in blood of protein, amino acids, ketone bodies, carnitine, glucose, galactose, lactate, insulin, and the electrolytes calcium, sodium, potassium and chloride; (c) the turnover of glucose and 3-hydroxybutyrate; (d) the concentration in brain of protein, cholesterol, cerebroside sulphate and the activities of the enzymes pyruvate dehydrogenase (EC 1.2.4.1), 3-oxo-acid-CoA transferase (EC 2.8.3.5) and acetoacetyl-CoA ligase (EC 6.2.1.16). 8. The studies suggest that milk-substitutes approximating to rats milk in composition promote acceptable metabolism in the artificially reared rat pup.

STAT3-mediated astrogliosis protects myelin development in neonatal brain injury.

Pathological findings in neonatal brain injury associated with preterm birth include focal and/or diffuse white matter injury (WMI). Despite the heterogeneous nature of this condition, reactive astrogliosis and microgliosis are frequently observed. Thus, molecular mechanisms by which glia activation contribute to WMI were investigated.

The origin of palmitic acid in brain of the developing rat

A rat milk substitute containing lower amounts of palmitic and oleic acid in the triacylglycerols in comparison to natural rat milk was fed to artificially reared rat pups from day 7 after birth to day 14. Pups reared by their mother served as controls. Free trideuterated (D3) palmitic acid [(C2H3)(CH2)14COOH, 98 atom % D] and free perdeuterated (D31) palmitic acid [C152H31COOH, 99 atom % D] in equal quantity were mixed into the triacylglycerols of the milk substitute in an amount equal to 100% of the palmitic acid in the triacylglycerols. A control milk substitute contained unlabeled free palmitic acid in an amount equal to 100% of the palmitic acid in the triacylglycerols of the milk substitute. The objective was to determine if palmitic acid in the diet contributed significantly to the palmitic acid content of developing brain and other organs. The methyl esters of the fatty acids were analyzed by gas chromatography and the palmitic acid methyl ester was examined by fast atom bombardment mass spectrometry. The proportion of deuterated methyl palmitate as a percentage of total palmitate was determined; 32% of the palmitic acid in liver and 12% of the palmitic acid in lung were trideuterated and perdeuterated palmitic acid in approximately equal amounts. The brain, by contrast, did not contain the deuterated palmitic acid moiety. Quantitation of palmitic acid and total fatty acids revealed a significant accumulation in organs in the interval from 7 to 14 days of age. Under our experimental conditions, labeled palmitic acid does not enter the brain. Consequently, we conclude that the developing brain produces all required palmitic acid byde novo synthesis.

Mild carbon monoxide exposure and auditory function in the developing rat.

We have examined the influence of chronic mild exposure to carbon monoxide (CO) on cognitive (learning) and auditory function in the developing rat. We have demonstrated that the auditory pathway is compromised at exposures less than 50 ppm, whereas learning was not influenced at 100 ppm. Artificially reared rat pups were exposed to CO during the brain growth spurt and onset of myelination. Spatial learning was assessed using the Morris Water Maze and three tests of auditory function: (1) auditory brainstem conduction times; (2) the amplitude of the eighth nerves action potential; and (3) otoacoustic emissions carried out on rat pups (age 22– 24 days). The pups were gastrostomy‐reared on a rat milk substitute and chronically exposed to CO at discrete concentrations in the range of 12–100 ppm from 6 days of age to post‐weaning at 21–23 days of age. We found no difference in auditory brainstem conduction times at all CO concentrations in comparison to non‐exposed controls. There was a difference in otoacoustic emissions for test and controls at CO concentrations of 50 ppm but not at lower concentrations. There was a consistent attenuation of the amplitude of the eighth nerves action potential, even at the lowest CO exposure examined. The attenuation of the amplitude of the action potential of the eighth nerve at 50 ppm carbon monoxide exposure did not completely recover by 73 days of age. We conclude that prolonged mild exposure to carbon monoxide during development causes measurable functional changes at the level of the eighth cranial nerve.

Growth and development of brain of artificially reared hypoketonemic rat pups

Abstract Three groups of rats were reared: mother-reared controls; artificially reared controls (AR-c), which were fed a milk substitute with the same composition of macronutrients as natural rats milk; and an artificially reared test group (AR-h), which was fed a milk substitute identical to that fed AR-c pups except that the component of fat containing medium chain length fatty acids was omitted (medium chain triglyceride deficient) and replaced on an isocaloric basis with carbohydrate. The AR rats were fed the milk substitute from postnatal Day 5 until Day 17 by fitting them with gastric cannulas through which the milk could be infused automatically. The nutritional impact of the milk substitutes on growth and the integrity of the brain was assessed by a comparison of morphologic and biochemical markers. Pups in the AR-h group were hypoketonemic. Animals in all groups attained the same body weight by Day 17 and there was no difference in the morphologic markers among the groups with one exception: the vibrissal “barrel fields” of the somatosensory cortex of rat pups in both AR groups were reduced in size but not in number or distribution from those of the mother-reared groups. Furthermore, the brains of the rat pups in the AR groups were not different in weight, but they weighed less than brains of mother-reared controls. Our data show that although there are many similarities in the status of AR rat pups when compared with motherreared controls, distinctive differences associated with artificial rearing are evident. We conclude that medium chain fatty acids in milk fat and the circulating ketone bodies are not mandatory substrates for growth and the development of the brain. Mechanisms must exist whereby alternative substrates are used to compensate when these metabolites are diminished in supply.

Mild carbon monoxide exposure impairs the developing auditory system of the rat

The object of this study was to determine if chronic exposure to mild concentrations of CO in air caused changes in the integrity of the inferior colliculus during the most active period of synaptogenesis/auditory development. We examined all subregions of the inferior colliculus (IC) of rats by immunocytochemical approaches after pups were exposed chronically to CO concentrations of, 0, 12.5, 25, and 50 ppm in air starting at Day 8 through 20–22 days of age. Mother‐reared pups were compared to the gastrostomy‐reared pups with or without CO exposure for basal neural activity, using c‐Fos immunoreactivity as a marker. Half the rats were examined at 27 days of age, 5 days after the end of CO exposure, and the other half were examined 50 days later at 75–77 days of age. In the central nucleus of the IC, the number of cells expressing a basal level of c‐Fos was decreased significantly in the CO‐exposed animals when compared to controls; however, there was little or no difference in the number of cells expressing c‐Fos in the other subregions of the IC. We conclude that the central nucleus of the inferior colliculus is affected selectively by mild CO exposure (0.0012% in air) and that this reduction in neuronal activity persists into adulthood.

Mild carbon monoxide exposure diminishes selectively the integrity of the cochlea of the developing rat

Rat pups were chronically exposed to carbon monoxide (CO) concentrations (12 or 25 ppm) in air starting at day 8, through 22 days of age, to examine the changes in the peripheral auditory system. Gastrostomy‐reared rat pups, with or without CO exposure, were used and compared with mother‐reared pups. The organ of Corti and the neurons of the spiral ganglion were analyzed for their morphology by using immunochemical and histological techniques. The inner and outer hair cells in the organ of Corti of animals exposed to 12 and 25 ppm CO were not different from the controls. However, at 25 ppm CO exposure, the nerve terminals innervating the inner hair cells were swollen. The somata of neurons in the spiral ganglion showed mild changes in the cytoplasm, and signs of mild vacuolization were observed in myelin covering their central processes. Synaptophysin, a marker for synaptic vesicles, and choline acetyltransferase, a marker for cholinergic terminals, showed no difference in immunoreactivity in CO exposed animals at 12 and at 25 ppm when compared with their age‐matched controls. Also, Na+K+ ATPase immunoreactivity patterns were normal compared with controls. Three enzymes were significantly reduced at the 25 ppm CO exposure: Cytochrome oxidase, NADH‐TR, and calcium ATPase were decreased in both the organ of Corti and the neurons of the spiral ganglion, and decreased immunostaining for the neurofilament and myelin basic proteins was found. We conclude that components of the cochlea are selectively affected by mild chronic CO exposure during development.