Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5865-5869, June 1995 Neurobiology A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury CHRISTOPHER G. WINTER*, YAS SAOTOME*, STEVEN W. LEVISONt, AND DAVID HIRSH* *Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; and tDepartment of Neuroscience and Anatomy, M. S. Hershey Medical Center, Hershey, PA 17033 Communicated by Richard Axel, College ofPhysicians and Surgeons of Columbia University, January 10, 1995 ABSTRACT Within the central nervous system (CNS) the cntf coding region and 2.3 kb of endogenous upstream ciliary neurotrophic factor (CNTF) is expressed by astrocytes sequence. The 2.3-kb upstream sequence spans the entire where it remains stored as an intracellular protein; its release intergenic distance between cntf and the gene immediately and function as an extracellular ligand are thought to occur preceding it, which is known as pzf (16). Therefore, it is likely in the event of cellular injury. We find that overexpression of that the transgene construct contains all of the endogenous CNTF in transgenic mice recapitulates the glial response to CNTF promoter. The transgene was microinjected into fertil- CNS lesion, as does its injection into the uninjured brain. ized eggs from CBA/C57BL mice. Eggs were subsequently These results demonstrate that CNTF functions as an inducer reimplanted into pseudopregnant foster mothers by standard of reactive gliosis, a condition associated with a number of techniques (17). Genomic DNA from biopsies of the tails of neurological diseases of the CNS. the offspring were analyzed to identify mice carrying the cntf transgene. Three single-locus transgenic mouse lines were Ciliary neurotrophic factor (CNTF) promotes the survival of established, each with a single distinct site of transgene a variety of neuronal populations in culture (1-4). It also acts integration. The number of transgene copies harbored by each as a differentiation and trophic factor on glial cells in vitro (5, line was determined by densitometric scanning of genomic 6). In vivo, exogenously added CNTF prevents the death of Southern blots. In all comparative studies, heterozygous trans- damaged neurons in several injury models (7, 8) and inhibits genic mice were compared to their wild-type litter mates. Mice the onset of symptoms associated with progressive motor were analyzed between 8 and 16 weeks of age. neuron degeneration in the pmn mutant mouse (9). The CNTF and Glial Fibrillary Acidic Protein (GFAP) Expres- function of CNTF as a ligand is thought to be limited to sion in Transgenic Tissue. The level of cntf transcription was instances that involve cellular injury because CNTF is a measured by scanning densitometry of the 1.2-kb mRNA nonsecreted cytosolic protein that lacks a consensus secretory species on a Northern blot autoradiograph. cntfmRNA levels signal sequence (10-12). However, the physiological role of are reported relative to wild-type brain (excluding olfactory CNTF upon injury remains unknown. bulb and optic nerve) and normalized by using mRNA for Ablation of the murine cntf gene has revealed an essential glyceraldehyde phosphate dehydrogenase (GAPDH) as an role for CNTF in postdevelopmental motor neuron mainte- internal standard. CNTF protein levels were determined in nance (13); CNTF-null mice do not begin to show an abnormal whole brain, including olfactory bulb, by using a chicken phenotype until adulthood, when they exhibit atrophy and loss sympathetic chain neuronal survival assay (18). Recombinant of motor neurons and diminished muscle strength. Based on human (rh) CNTF was used as a standard to calculate the these results, it has been proposed that microtrauma caused by amount of CNTF present in the mouse tissue extracts. The normal physical stress permits the release of CNTF into the levels of mRNA for GFAP were determined by Northern blot extracellular environment where it exerts its trophic action in analysis of total RNA isolated from the olfactory bulbs of maintaining motor neuron viability. wild-type or transgenic mice. A 540-bp Sca I-Spe I fragment CNTF has been shown to influence the differentiation of of the rat gfap cDNA was used as a hybridization probe. glial precursor cells in vitro (5, 6); however, evidence for an Quantification ofband intensitywas performed with a Packard ontogenetic role in this process in vivo is lacking. By using InstantImager. Values were corrected for differences in the transgenic mice that overexpress CNTF, we assessed the amounts of total RNA loaded by using mRNA levels for possible function of CNTF in the development of astrocytes GAPDH. gap mRNA was measured in three samples of and oligodendrocytes. We find that mice that overexpress wild-type and transgenic olfactory bulb total RNA and found CNTF display no overt developmental glial abnormalities. to be similar in all samples. However, elevated levels of CNTF in transgenic mice lead to Immunohistochemistry and Carnosine Determination. the induction of a glial phenotype, reactive gliosis, that is Adult mice were perfused with 4% (wt/vol) paraformalde- characteristically observed in cases of central nervous system hyde; olfactory bulbs were removed, postfixed in 4% paraform- (CNS) injury and disease (14, 15). Further, exogenous CNTF aldehyde, incubated in 30% (wt/vol) sucrose, snap-frozen, and supplied by intracerebral injection also led to gliosis in normal embedded for sectioning. Spinal cords were dissected from mice. These results support the proposal that a function of perfused animals after in vivo postfixation overnight at 4°C and CNTF in the CNS may be in regulating the glial response to then embedded in freezing medium. A cryostat microtome was neural lesion. used to cut 10-,tm coronal sections of the olfactory bulbs and 20-,tm longitudinal sections of the spinal cords. Sections were MATERIALS AND METHODS mounted on gelatin-coated glass microscope slides. Sections were treated with 0.3% hydrogen peroxide and incubated Production of cntf Transgenic Mice. Transgenic mice were sequentially with phosphate-buffered saline (PBS), mouse generated by using 5.3 kb of murine genomic DNA, including anti-GFAP monoclonal antibody (Boehringer Mannheim) di- The publication costs of this article were defrayed in part by page charge Abbreviations: CNS, central nervous system; CNTF, ciliary neurotro- payment. This article must therefore be hereby marked "advertisement" in phic factor; GFAP, glial fibrillary acidic protein; OMP, olfactory accordance with 18 U.S.C. §1734 solely to indicate this fact. marker protein; rh, recombinant human. 5865 Downloaded by guest on September 24, 2021 5866 Neurobiology: Winter et al Proc. Natl. Acad. Sci. USA 92 (1995) luted 1:50 in PBS containing 1% normal goat serum or rat GFAP staining in the wild-type olfactory bulb was relatively anti-Mac-1 monoclonal antibody (Boehringer Mannheim) di- weak and largely confined to the glomerular region (Fig. 1B). luted 1:100, twice with 1% normal goat serum, and either In the transgenic mice, GFAP+ cells were present throughout biotin-labeled goat anti-mouse IgG F(ab')2 fragment (for the olfactory bulb with densely stained plaques of GFAP found GFAP) or biotin-labeled goat anti-rat IgG F(ab')2 fragment in the glomeruli (Fig. 1A). Astrocytes of the transgenic (for Mac-1). Olfactory marker protein (OMP) staining was olfactory bulb were hypertrophied and had thick intensely carried out as described above, except that goat anti-OMP was staining GFAP+ processes (Fig. 1D) characteristic of reactive used at a dilution of 1:5000 and a biotin-conjugated rabbit astrocytes associated with insult and disease in the CNS (14, anti-goat F(ab')2 fragment was used as a secondary antibody. 15). This contrasts with the appearance of the astrocytes in An ABC peroxidase detection system (Vector Laboratories) wild-type mice (Fig. 1E). Elevation in GFAP staining was also was used for the final staining steps. Olfactory bulb carnosine observed in the olfactory bulb of another CNTF transgenic line levels were measured by a spectrophotometric method (19). (t3), indicating that this phenotype is independent of the locus Experimental values were extrapolated from a standard curve of transgene integration. generated by using pure carnosine (Sigma). In the CNS, the Mac-1 antigen (CD11b) is expressed by brain Induction of Neuronal Degeneration. To induce sensory macrophages and microglia (23). These phagocytic scavenger neuron degeneration, wild-type mice were treated intranasally cells are a prominent feature of reactive gliosis in areas of CNS with ZnSO4 as described (20). lesion (24). Staining with an antibody against Mac-1 revealed the Stereotactic Injection of CNTF. CNTF protein (0.36 ,g of presence of microglia/brain macrophages in the olfactory bulbs rhCNTF) was administered intracerebrally by using a ster- of the cntf transgenic mice, specifically within the glomerular eotactic injection apparatus. Heat-treated CNTF was used as layer (Fig. iF). The olfactory bulbs ofwild-type mice were devoid a negative control. It was shown independently to have no of Mac-1+ cells (Fig. 1G). In summary, the bulbs of transgenic biological activity in the chicken sympathetic chain neuronal mice overexpressing CNTF exhibit three salient characteristics of survival assay (18). In the same assay, the native rhCNTF used reactive gliosis: induction of GFAP, hypertrophy of astrocytes, in this