Florencia Labombarda

Progesterone neuroprotection in traumatic CNS injury and motoneuron degeneration

Studies on the neuroprotective and promyelinating effects of progesterone in the nervous system are of great interest due to their potential clinical connotations. In peripheral neuropathies, progesterone and reduced derivatives promote remyelination, axonal regeneration and the recovery of function. In traumatic brain injury (TBI), progesterone has the ability to reduce edema and inflammatory cytokines, prevent neuronal loss and improve functional outcomes. Clinical trials have shown that short-and long-term progesterone treatment induces a significant improvement in the level of disability among patients with brain injury. In experimental spinal cord injury (SCI), molecular markers of functional motoneurons become impaired, including brain-derived neurotrophic factor (BDNF) mRNA, Na,K-ATPase mRNA, microtubule-associated protein 2 and choline acetyltransferase (ChAT). SCI also produces motoneuron chromatolysis. Progesterone treatment restores the expression of these molecules while chromatolysis subsided. SCI also causes oligodendrocyte loss and demyelination. In this case, a short progesterone treatment enhances proliferation and differentiation of oligodendrocyte progenitors into mature myelin-producing cells, whereas prolonged treatment increases a transcription factor (Olig1) needed to repair injury-induced demyelination. Progesterone neuroprotection has also been shown in motoneuron neurodegeneration. In Wobbler mice spinal cord, progesterone reverses the impaired expression of BDNF, ChAT and Na,K-ATPase, prevents vacuolar motoneuron degeneration and the development of mitochondrial abnormalities, while functionally increases muscle strength and the survival of Wobbler mice. Multiple mechanisms contribute to these progesterone effects, and the role played by classical nuclear receptors, extra nuclear receptors, membrane receptors, and the reduced metabolites of progesterone in neuroprotection and myelin formation remain an exciting field worth of exploration.

Progesterone up-regulates neuronal brain-derived neurotrophic factor expression in the injured spinal cord.

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Recent studies point to neurotrophins as possible mediators of hormone action. Here, we show that the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels was increased by PROG treatment in ventral horn motoneurons from rats with spinal cord injury (SCI). Semiquantitative in situ hybridization revealed that SCI reduced BDNF mRNA levels by 50% in spinal motoneurons (control: 53.5+/-7.5 grains/mm(2) vs. SCI: 27.5+/-1.2, P<0.05), while PROG administration to injured rats (4 mg/kg/day during 3 days, s.c.) elicited a three-fold increase in grain density (SCI+PROG: 77.8+/-8.3 grains/mm(2), P<0.001 vs. SCI). In addition, PROG enhanced BDNF immunoreactivity in motoneurons of the lesioned spinal cord. Analysis of the frequency distribution of immunoreactive densities (chi(2): 812.73, P<0.0001) showed that 70% of SCI+PROG motoneurons scored as dark stained whereas only 6% of neurons in the SCI group belonged to this density score category (P<0.001). PROG also prevented the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. In the normal intact spinal cord, PROG significantly increased BDNF inmunoreactivity in ventral horn neurons, without changes in mRNA levels. Our findings suggest that PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection.

Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors, and myelin proteins following spinal cord injury

Progesterone is emerging as a myelinizing factor for central nervous system injury. Successful remyelination requires proliferation and differentiation of oligodendrocyte precursor cells (OPC) into myelinating oligodendrocytes, but this process is incomplete following injury. To study progesterone actions on remyelination, we administered progesterone (16 mg/kg/day) to rats with complete spinal cord injury. Rats were euthanized 3 or 21 days after steroid treatment. Short progesterone treatment (a) increased the number of OPC without effect on the injury‐induced reduction of mature oligodendrocytes, (b) increased mRNA and protein expression for the myelin basic protein (MBP) without effects on proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), and (c) increased the mRNA for Olig2 and Nkx2.2 transcription factors involved in specification and differentiation of the oligodendrocyte lineage. Furthermore, long progesterone treatment (a) reduced OPC with a concomitant increase of oligodendrocytes; (b) promoted differentiation of cells that incorporated bromodeoxyuridine, early after injury, into mature oligodendrocytes; (c) increased mRNA and protein expression of PLP without effects on MBP or MOG; and (d) increased mRNA for the Olig1 transcription factor involved in myelin repair. These results suggest that early progesterone treatment enhanced the density of OPC and induced their differentiation into mature oligodendrocytes by increasing the expression of Olig2 and Nkx2.2. Twenty‐one days after injury, progesterone favors remyelination by increasing Olig1 (involved in repair of demyelinated lesions), PLP expression, and enhancing oligodendrocytes maturation. Thus, progesterone effects on oligodendrogenesis and myelin proteins may constitute fundamental steps for repairing traumatic injury inflicted to the spinal cord.

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.

Progesterone Prevents Allodynia After Experimental Spinal Cord Injury

UNLABELLED Chronic pain after spinal cord injury represents a therapeutic challenge. Progesterone, a neuroprotective steroid, has been shown to modulate nociceptive thresholds, whereas its effect on neuropathic pain needs to be further explored. In this study, we evaluated whether progesterone could ameliorate pain-associated behaviors in animals subjected to a spinal cord hemisection. The development of mechanical and cold allodynia was assessed in injured male rats treated with daily injections of progesterone or vehicle. The expression of N-methyl-D-aspartate receptor (NMDAR) subunits, protein kinase C gamma (PKCγ), preprodynorphin (ppD), and kappa opioid receptor (KOR), key players in chronic pain mechanisms, was determined in the dorsal spinal cord. Twenty-eight days after injury, all vehicle-treated animals presented allodynic behaviors and a marked increase in NMDAR subunits, PKCγ, and ppD mRNA levels, with no changes in KOR mRNA levels. Progesterone prevented the development of mechanical allodynia and reduced the painful responses to cold stimulation. In correlation with the attenuation of pain behaviors, the steroid prevented NMDAR subunits and PKCγ mRNAs upregulation, did not modify the elevated ppD mRNA levels, but increased KOR expression. In conclusion, progesterone modulates neuropathic pain after spinal cord injury, creating a favorable molecular environment that may decrease spinal nociceptive signaling. PERSPECTIVE The present study suggests that progesterone administration could represent an interesting strategy to modulate neuropathic pain circuits after spinal cord injury. Further studies are needed to investigate the potential progesterone receptors involved in these actions.

Progesterone prevents nerve injury-induced allodynia and spinal NMDA receptor upregulation in rats.

BACKGROUND Peripheral nerve injury-evoked neuropathic pain still remains a therapeutic challenge. Recent studies support the notion that progesterone, a neuroactive steroid, may offer a promising perspective in pain modulation. OBJECTIVES Evaluate the effect of progesterone administration on the development of neuropathic pain-associated allodynia and on the spinal expression of N-Methyl-D-Aspartate Receptor subunit 1 (NR1), its phosphorylated form (pNR1), and the gamma isoform of protein kinase C (PKCγ), all key players in the process of central sensitization, in animals subjected to a sciatic nerve constriction. METHODS Male Sprague-Dawley rats were subjected to a sciatic nerve single ligature constriction and treated with daily subcutaneous injections of progesterone (16 mg/kg) or vehicle. The development of hindpaw mechanical and thermal allodynia was assessed using the von Frey and Choi tests, respectively. Twenty two days after injury, the number of neuronal profiles exhibiting NR1, pNR1, or PKCγ immunoreactivity was determined in the dorsal horn of the lumbar spinal cord. RESULTS Injured animals receiving progesterone did not develop mechanical allodynia and showed a significantly lower number of painful responses to cold stimulation. In correlation with the observed attenuation of pain behaviors, progesterone administration significantly reduced the number of NR1, pNR1, and PKCγ immunoreactive neuronal profiles. CONCLUSIONS Our results show that progesterone prevents allodynia in a rat model of sciatic nerve constriction and reinforce its role as a potential treatment for neuropathic pain.

Neuropathic pain and temporal expression of preprodynorphin, protein kinase C and N-methyl-d-aspartate receptor subunits after spinal cord injury

Central neuropathic pain is refractory to conventional treatment and thus remains a therapeutic challenge. In this work, we used a well-recognized model of central neuropathic pain to evaluate time-dependent expression of preprodynorphin (ppD), protein kinase C gamma (PKCgamma) and NMDA receptor (NMDAR) subunits NR1, NR2A and NR2B, all critical players in nociceptive processing at the spinal level. Male Sprague-Dawley rats were subjected to spinal hemisection at T13 level and sham-operated rats were included as control animals. The development of hindpaw mechanical allodynia was assessed using the von Frey filaments test. Real time RT-PCR was employed to determine the relative mRNA levels of NMDAR subunits, ppD and PKCgamma in the dorsal spinal cord 1, 14 and 28 days after injury. Our results show that, coincident with the allodynic phase after injury, there was a strong up-regulation of the mRNAs coding for ppD, PKCgamma and NMDAR subunits in the dorsal spinal cord caudal to the injury site. The present study provides further evidence that these molecules are involved in the development/maintenance of central neuropathic pain and thus could be the target of therapeutic approaches.

Progesterone reduces the expression of spinal cyclooxygenase‐2 and inducible nitric oxide synthase and prevents allodynia in a rat model of central neuropathic pain

Spinal cord injury (SCI) results in the development of chronic pain that is refractory to conventional treatment. Progesterone, a neuroprotective steroid, may offer a promising perspective in pain modulation after central injury. Here, we explore the impact of progesterone administration on the post‐injury inflammatory cascade involving the enzymes cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) at the spinal cord level. We also analyse pain behaviours, the profile of glial cell activation, and IκB‐α mRNA levels, as an index of NF‐κB transactivation.

Glucocorticoid effects on Fos immunoreactivity and NADPH-diaphorase histochemical staining following spinal cord injury.

Glucocorticoids (GC) provide neuroprotection and early recovery after spinal cord injury (SCI). While several mechanisms were proposed to account for these effects, limited information exists regarding GC actions in sensory areas of the spinal cord. Presently, we studied the time course of Fos expression, and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical staining to monitor neuronal responses to SCI with or without GC treatment. Rats with sham-operation or transection at the thoracic level (T7-T8) received vehicle or 5 mg/kg of the GC dexamethasone (DEX) at 5 min post-lesion and were sacrificed 2 or 4 h after surgery. Another group of SCI rats received vehicle or intensive DEX treatment (5 min, 6 h, 18 h and 46 h post-lesion) and were sacrificed 48 h after surgery. The number of NADPH-d positive neurons or Fos immunoreactive nuclei was studied by computer-assisted image analysis in superficial dorsal horn (Laminae I-III) and central canal area (Lamina X) below the lesion. While constitutive Fos immunoreactive nuclei were sparse in controls, SCI increased Fos expression at 2 and 4 h after injury. DEX treatment significantly enhanced the number of Fos positive nuclei in Laminae I-III by 4 h after transection, although the response was not maintained by intensive steroid treatment when tested at 48 h after SCI. NADPH-d positive neurons in Laminae I-III increased at 2 and 4 h after SCI while a delayed increased was found in central canal area (Lamina X). DEX treatment decreased NADPH-d positive neurons to sham-operated levels at all time points examined. Thus, while GC stimulation of Fos suggests activation of neurons involved in sympathetic outflow and/or pain, down-regulation of NADPH-d indicates attenuation of nociceptive outflow, considering the role of enzyme-derived nitric oxide in pain-related mechanisms. Differential hormonal effects on these molecules agree with their localization in different cell populations.

Progesterone modulates pro-inflammatory cytokine expression profile after spinal cord injury: Implications for neuropathic pain

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. Glial cell activation and cytokine production contribute to the pathology of central neuropathic syndromes. In this study we evaluated the effects of progesterone, a neuroactive steroid, on pain development and the spinal expression of IL-1β, its receptors (IL-1RI and IL-1RII) and antagonist (IL-1ra), IL-6 and TNFα, and NR1 subunit of NMDAR. Our results show that progesterone, by modulating the expression of pro-inflammatory cytokines and neuronal IL-1RI/NR1 colocalization, emerges as a promising agent to prevent chronic pain after SCI.