This work examines whether microglia-conditioned medium (MCM) is beneficial in stressed spinal cord cells or tissues. MCM was separated into two fractions by 50 kDa molecular cut-off centrifugation. MCM not only promoted survival of neuronal and oligodendroglial cells but effectively reduced LPS stimulation in spinal cord cultures. We further utilized the NYU weight-drop device to induce contusive spinal cord injury (SCI) in rats. Immediately after dropping the impactor from a height of 25 mm onto thoracic spinal segment, MCM was intrathecally administered. At 6 weeks post-injury, SCI rats receiving MCM > 50 kDa treatment showed significant hind-limb improvement over MCM < 50 kDa- or vehicle-treated SCI rats. Consistently, more preserved nerve fibers and fewer activated microglia were found in the injured epicenter of MCM-treated SCI rats. Taken together, secreted substances, mainly > 50 kDa, of microglia was neuroprotective against spinal cord injury.
Microglial cells are immunoreactive cells of nonneural lineage resident in the CNS and play an important role in the physiological and pathological conditions of the brain. After injury to the CNS, microglia are rapidly activated and concentrated at the sites of injury. Several studies have shown that activated microglia attribute to destructtive processes leading to secondary neuronal degeneration. However, activated microglia also expresses neuroprotective and neurotrophic activities [1-3]. A better understanding of the role of microglia and of inflammation in brain/spinal cord injuries is required to develop treatments to prevent nerve damage and improve repair.
Traumatic spinal cord injury (SCI) is an unexpected and devastating event which leads to loss of neurological function below the level of injury. SCI disrupts long projection axons and initiates a series of primary and secondary injury cascades. SCI elicits an inflammatory response that recruits macrophages to the injured spinal cord. Microglia/macrophage can become over-activated under certain circumstances and produce an excess of cytotoxic factors like superoxide, nitric oxide, and tumor necrosis factor-a (TNF-a) [
A prominent role for secreted molecules in microglia has been suggested by numerous studies; however, few studies have reports or direct evidence on spinal cord. In the present work we tested whether microglia-conditioned medium (MCM) could protect spinal cord from contusive injury. Here we show that MCM was beneficial in spinal cord cultures. Furthermore, when MCM was intrathecally injected to spinal cord after SCI, neurological recovery was improved and spinal cord pathology was reduced.
Lipopolysaccharide (LPS; Escherichia coli O111:B4) was purchased from Sigma-Aldrich (St. Louis, USA). Culture multi-wells and pipettes were obtained from Orange Scientific (Graignette, Belgium). Cultured media, fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Invitrogen Corporation, USA). Other reagents were purchased from Sigma-Aldrich unless stated otherwise.
Spinal cord neuronal/glial cultures were prepared from embryonic Sprague-Dawley (SD) rat spinal cords at gestation days 14 - 16 as described in Hung [
Medium of enriched microglia was replaced with fresh medium. Two days later, the conditioned media was collected, cleared from free-floating cells by centrifugation for 5 min at 1000 × g, and sterile filtered. For fractionation of conditioned medium, MCM was further passed through an Amicon Ultracentrifugal filter with 50 kDa cut-off (Millipore, USA) by centrifugation 3000 g at 4˚C. The resulted two fractions (>50 kDa or <50 kDa) of MCM were saved and frozen until use. For cell survival test, spinal cord neuronal/glial cultures were maintained in the presence or absence of MCM for 5 days and then fixed for immunostaining. For examining the effect of LPS stimulation, cultures were treated with MCM in the presence or absence of LPS (1.2 µg/ml) on the 2nd or 3rd day after seeding. Two days later, cultured medium was collected for Griess assay of released nitrite, an indicator of nitric oxide, and the cells were fixed for immunostaining.
SCI was induced in adult SD rats using the New York University weight-drop device as described previously [
Rats were allowed to recover for 6 weeks post-injury. This time course allowed for examining the effect of MCM during the initial deficit in the behavioral locomotor assay and during the plateau phase of recovery after trauma. Recovery of hindlimb stepping was assessed while subjects moved freely about an open field. The Basso, Beattie and Bresnehan (BBB) open field score was used to evaluate locomotion in terms of hindlimb functional improvement of the SCI rats as previously described [
NO production was assessed using the Griess reaction. The production of NO was assayed as the accumulation of nitrite in the medium using Griess reagents (1% sulfanilaminde/0.1% naphthylethylene diamine dihydrochloride/2% phosphoric acid) as described by Tsai et al. [
Cultured cells were fixed in 4% paraformaldehyde at room temperature for 30 min. Cells were further permeabilized with 0.2% Triton X-100, blocked with 2% serum and immunostained with primary antibodies and with the respective fluorescently tagged secondary antibodies (Jackson ImmunoResearch Inc.). Primary antibodies included mouse anti-inducible nitric oxide synthase (iNOS) (BD Bioscience, USA), rabbit anti-bIII tubulin (covance, USA) and rabbit anti-MBP (Chemicon, USA). Images of cultured cells were obtained with a fluorescent microscope equipped with fluorescence optics and with a CCD camera. Animals were perfused with 4% paraformaldehyde at 6 weeks postinjury. The spinal cords (approximately 2 mm length with the contused site at the center of the sample) were removed and processed for longitudinal sectioning at a thickness of 20 mm. Immunostaining was performed as described previously [
Experimental data were expressed as the mean of independent values ± S.E.M. One-way ANOVA followed by Bonferroni’s t-test was used to determine statistical differences between treatments. A statistically significant difference was accepted at P < 0.05.
Enriched microglial cells were purified from mixed glial cultures. As shown in Figures 1(A) and (B), more than 99% of cells were immunoreactive to ED-1, a microglial marker. MCM was collected from such enriched microglia. Spinal cord neuronal/glial cultures were treated with MCM or PBS, as a vehicle control, for 5 days. Cultures were then processed for immunohistochemical staining.
More neurite connections of bIII were observed in MCM-treated cells (
The hindlimb motor function of injured rats receiving various treatments was monitored according to the guideline of BBB Locomotor Rating Scale [16,17]. As shown in
NF-L-IR fibers (
The central observation of the present study is that MCM was beneficial/anti-inflammatory to spinal cord cultures as well as to injured spinal cords in vivo. This conclusion is supported by the following evidence. First, MCM not only enhanced neuronal survival/connection, but also increased the processes of mature oligodendroglia in spinal cord cultures. Second, MCM effectively attenuated LPS stimulation in cultures. Third, intrathecal injection of MCM significantly improved functional restoration in SCI rats in vivo. Fourth, recovery in hindlimb locomotor function induced by MCM (>50 kDa fraction) appeared to be correlated with the nerve fibers remained in the injured spinal cord. Fewer infiltrated ED-1 (+) microglia/ macrophages were found in the injured cord of MCMtreated SCI rats. Accordingly, the effect of MCM on functional recovery could be due to preventing neurons from insult and inflammation caused by SCI. Our results support the concept that microglia exert neuroprotective effects mainly through the release of soluble factors.
Increased neuronal and oligodendroglial connection by MCM was demonstrated by higher density of bIII-tubulinIR, SV2-IR and MBP-IR in MCM-treated cultures. Tubulin makes up microtubules. SV2-IR-synaptic vesicles are distributed within presynaptic terminals. The result indicated a dendritic and axonal promoting effect of MCM. MBP, a major structural protein in myelin, is responsible primarily for compaction and stabilization of the major dense line [
Several lines of evidence have demonstrated that microglia could be neuroprotective after ischemia [21-25]. Yu [
As a conclusion remark, results of the present study demonstrated a neuroprotective effect of MCM on injured spinal cord both in cultures and in vivo. These data contributed to the therapeutic use of microglia-derived products to improve repair in injured nervous system.
This work was supported by Grants V101E6-001, V100S6-001 and V99S6-001 from the Taipei Veterans General Hospital in Taiwan, by a Grant NSC 99-2628-B-010-009-MY3 from the National Science Council in Taiwan, and by a grant from the Ministry of Education (Aim for the Top University Plan).