Tg21 Mice Research Articles

Cerebral Erythropoietin Prevents Sex-Dependent Disruption of Respiratory Control Induced by Early Life Stress.

Injuries that occur early in life are often at the root of adult illness. Neonatal maternal separation (NMS) is a form of early life stress that has persistent and sex-specific effects on the development of neural networks, including those that regulate breathing. The release of stress hormones during a critical period of development contributes to the deleterious consequences of NMS, but the role of increased corticosterone (CORT) in NMS-induced respiratory disturbance is unknown. Because erythropoietin (EPO) is a potent neuroprotectant that prevents conditions associated with hyperactivation of the stress neuroaxis in a sex-specific manner, we hypothesized that EPO reduces the sex-specific alteration of respiratory regulation induced by NMS in adult mice. Animals were either raised under standard conditions (controls) or exposed to NMS 3 h/day from postnatal days 3–12. We tested the efficacy of EPO in preventing the effects of NMS by comparing wild-type mice with transgenic mice that overexpress EPO only in the brain (Tg21). In 7-days-old pups, NMS augmented CORT levels ~2.5-fold by comparison with controls but only in males; this response was reduced in Tg21 mice. Respiratory function was assessed using whole-body plethysmography. Apneas were detected during sleep; the responsiveness to stimuli was measured by exposing mice to hypoxia (10% O2; 15 min) and hypercapnia (5% CO2; 10 min). In wild-type, NMS increased the number of apneas and the hypercapnic ventilatory response (HcVR) only in males; with no effect on Tg21. In wild-type males, the incidence of apneas was positively correlated with HcVR and inversely related to the tachypneic response to hypoxia. We conclude that neural EPO reduces early life stress-induced respiratory disturbances observed in males.

Cerebral erythropoietin promotes sex dependent prevention of disrupted neural control of respiration induced by neonatal stress

Stress experienced during a critical period of development is a major cause of adult disease (Kivimäki and Steptoe, 2018; Nelson and Gabard-Durnam, 2020; Shonkoff, 2016; Shonkoff et al., 2009); it has persistent and sex-specific effects on health and the brain is a major target. One neural system of considerable interest is the neural system regulating breathing. Pathology in this homeostatic control system underlies important clinical disorders such as sleep disordered breathing (SDB). Adult rodents previously subjected to early life stress in the form neonatal maternal separation (NMS) show key features of SDB, including a greater incidence of apneas during sleep and a significant sexual dimorphism. Erythropoietin (EPO) is a potent neuroprotective factor secreted by neurons and astrocytes that prevents stress-related neurological insults. Because treatment options for SDB are limited, we tested the hypothesis that over expression of neural EPO alleviates NMS-related respiratory control dysfunction in adult mice. To test this hypothesis, we used a transgenic animal model that selectively overexpress EPO in the brain (Tg21). Mouse pups were raised under standard conditions or subjected to NMS (3h/day; postnatal days 3 to 12). At adulthood, the apnea index and reflexive responses to O2 (hypoxia) and CO2 (hypercapnia) were measured using whole body plethysmography; corticosterone levels were measured by ELISA assay. In wild type, NMS augmented apnea frequency in males (not females). This effect of NMS was not observed in Tg21 mice. NMS also caused a sex-specific increase in basal corticosterone (males only). EPO overexpression reduced this effect. We conclude that overexpression of neural EPO prevents the deleterious impacts of NMS on the development of the stress neuraxis and respiratory control.

Erythropoietin Stimulates GABAergic Maturation in the Mouse Hippocampus.

Several neurodevelopmental disabilities are strongly associated with alterations in GABAergic transmission, and therapies to stimulate its normal development are lacking. Erythropoietin (EPO) is clinically used in neonatology to mitigate acute brain injury, and to stimulate neuronal maturation. Yet it remains unclear whether EPO can stimulate maturation of the GABAergic system. Here, with the use of a transgenic mouse line that constitutively overexpresses neuronal EPO (Tg21), we show that EPO stimulates postnatal GABAergic maturation in the hippocampus. We show an increase in hippocampal GABA-immunoreactive neurons, and postnatal elevation of interneurons expressing parvalbumin (PV), somatostatin (SST), and neuropeptide Y (NPY). Analysis of perineuronal net (PNN) formation and innervation of glutamatergic terminals onto PV+ cells, shows to be enhanced early in postnatal development. Additionally, an increase in GABAAergic synapse density and IPSCs in CA1 pyramidal cells from Tg21 mice is observed. Detection of EPO receptor (EPOR) mRNA was observed to be restricted to glutamatergic pyramidal cells and increased in Tg21 mice at postnatal day (P)7, along with reduced apoptosis. Our findings show that EPO can stimulate postnatal GABAergic maturation in the hippocampus, by increasing neuronal survival, modulating critical plasticity periods, and increasing synaptic transmission. Our data supports EPO's clinical use to balance GABAergic dysfunction.

Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.

Brain-derived erythropoietin protects from intermittent hypoxia-induced cardiorespiratory dysfunction and oxidative stress in mice.

Based on the fact that erythropoietin (Epo) administration in rodents protects against spatial learning and cognitive deficits induced by chronic intermittent hypoxia (CIH)-mediated oxidative damage, here we tested the hypothesis that Epo in the brain protects against cardiorespiratory disorders and oxidative stress induced by CIH in adult mice. Adult control and transgenic mice overexpressing Epo in the brain only (Tg21) were exposed to CIH (21%-10% O2-10 cycles/hour-8 hours/day-7 days) or room air. After CIH exposure, we used the tail cuff method to measure arterial pressure, and whole-body plethysmography to assess the frequency of apneic episodes at rest, minute ventilation, and ventilatory responses to hypoxia and hypercapnia. Finally, the activity of pro-oxidant (XO-xanthine oxidase, and NADPH) and antioxidant (super oxide dismutase) enzymes was evaluated in the cerebral cortex and brainstem. Exposure of control mice to CIH significantly increased the heart rate and arterial pressure, the number of apneic events, and the ventilatory response to hypoxia and hypercapnia. Furthermore, CIH increased the ratio of pro-oxidant to antioxidant enzymes in cortex and brainstem tissues. Both physiological and molecular changes induced by CIH were prevented in transgenic Tg21 mice. We conclude that the neuroprotective effect of Epo prevents oxidative damage in the brain and cardiorespiratory disorders induced by CIH. Considering that Epo is used in clinics to treat chronic kidney disease and stroke, our data show convincing evidence suggesting that Epo may be a promising alternative drug to treat sleep-disorder breathing.

Erythropoietin reduces experimental autoimmune encephalomyelitis severity via neuroprotective mechanisms

BackgroundTreatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo’s beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both?MethodsTo address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival.ResultsAlthough disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons.ConclusionsOur data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

Erythropoietin impacts the stress‐related respiratory dysfunction in mice: neonatal maternal separation and sex dimorphism

Neonatal maternal separation (NMS) is an established model of early life adversity that mimics stressful conditions experienced by newborn and infants. NMS disrupts brain development and as adults, individuals show sex‐dependent abnormal neuroendocrine response, predisposing them to physiological/psychological disorders. Concerning the neural control of respiration, studies in rats revealed that NMS causes respiratory instability and increased levels of corticosterone that remained during adulthood. Moreover, increased ventilatory response to hypoxia (HVR) but decreased to hypercapnia (HcVR) was observed in male animals, while decreased HVR but increased HcVR was observed in female rats.Although it is primarily recognized for its capacity to stimulate erythrocyte production, erythropoietin (Epo) is a cytokine that is also produced in the brain (by neurons and astrocytes), and plays role in development, maintenance, protection and repair of the nervous system. Studies from our lab demonstrated that cerebral Epo is a potent and sex‐specific respiratory stimulant that enhances the ability to tolerate hypoxic conditions at neonatal and adult ages. As cerebral Epo also attenuates the activation of the hypothalamic‐pituitary‐adrenal (HPA) axis, here we hypothesized that Epo protects against the stress‐induced cardio‐respiratory dysfunction produced by NMS. To test this hypothesis, circulating corticosterone level of adult (3 months) male and female Tg21 mice, was increased by subcutaneous implantation of beeswax pellets. 14 days later basal ventilation, HVR (10%O2 in N2) and HcVR (5% CO2, 21%O2 in N2) was evaluated by whole‐body plethysmography. Our preliminary results showed that corticosterone does not alter basal ventilation nor HVR in female and male Tg21 mice. However, corticosterone decreased HcVR in female Tg21 mice, meanwhile males showed an increased HcVR. These results suggest that Epo modulates the cortiscosterone‐induced cardiorespiratory dysfunctions.Support or Funding InformationCanadian Institutes of HealthResearch (MOP 130258), the Star Foundation for Children's HealthResearch. The salary of Jorge Soliz is supported by the “Fonds de recherche du Quebec‐Santé” (FRQ‐S).

Hypercapnic ventilatory response is decreased in a mouse model of excessive erythrocytosis.

The impact of cerebral erythropoietin (Epo) in the regulation of the hypercapnic ventilatory response (HcVR) is controversial. While we reported that cerebral Epo does not affect the central chemosensitivity in C57Bl6 mice receiving an intracisternal injection of sEpoR (the endogenous antagonist of Epo), a recent study in transgenic mice with constitutive high levels of human Epo in brain and circulation (Tg6) and in brain only (Tg21), showed that Epo blunts the HcVR, maybe by interacting with central and peripheral chemoreceptors. High Epo serum levels in Tg6 mice lead to excessive erythrocytosis (hematocrit ~80-90%), the main symptom of chronic mountain sickness (CMS). These latter results support the hypothesis that reduced central chemosensitivity accounts for the hypoventilation observed in CMS patients. To solve this intriguing divergence, we reevaluate HcVR in Tg6 and Tg21 mouse lines, by assessing the metabolic rate [O consumption (V̇) and CO production (V̇)], a key factor modulating ventilation, the effect of which was not considered in the previous study. Our results showed that the decreased HcVR observed in Tg6 mice (~70% reduction; < 0.01) was due to a significant decrease in the metabolism (~40%; < 0.0001) rather than Epo's effect on CO chemosensitivity. Additional analysis in Tg21 mice did not reveal differences of HcVR or metabolism. We concluded that cerebral Epo does not modulate the central chemosensitivity system, and that a metabolic effect upon CO inhalation is responsible for decreased HcVR observed in Tg6 animals. As CMS patients also show decreased HcVR, our findings might help to better understand respiratory disorders at high altitude.

Polycythemia and high levels of erythropoietin in blood and brain blunt the hypercapnic ventilatory response in adult mice.

Changes in arterial Po2, Pco2, and pH are the strongest stimuli sensed by peripheral and central chemoreceptors to adjust ventilation to the metabolic demand. Erythropoietin (Epo), the main regulator of red blood cell production, increases the hypoxic ventilatory response, an effect attributed to the presence of Epo receptors in both carotid bodies and key brainstem structures involved in integration of peripheral inputs and control of breathing. However, it is not known whether Epo also has an effect on the hypercapnic chemoreflex. In a first attempt to answer this question, we tested the hypothesis that Epo alters the ventilatory response to increased CO2 levels. Basal ventilation and hypercapnic ventilatory response (HCVR) were recorded from control mice and from two transgenic mouse lines constitutively expressing high levels of human Epo in brain only (Tg21) or in brain and plasma (Tg6), the latter leading to polycythemia. To tease apart the potential effects of polycythemia and levels of plasma Epo in the HCVR, control animals were injected with an Epo analog (Aranesp), and Tg6 mice were treated with the hemolytic agent phenylhydrazine after splenectomy. Ventilatory parameters measured by plethysmography in conscious mice were consistent with data from electrophysiological recordings in anesthetized animals and revealed a blunted HCVR in Tg6 mice. Polycythemia alone and increased levels of plasma Epo blunt the HCVR. In addition, Tg21 mice with an augmented level of cerebral Epo also had a decreased HCVR. We discuss the potential implications of these findings in several physiopathological conditions.

Cerebral erythropoietin regulates the respiratory control system during early postnatal life

Apart from its role in neuroprotection, erythropoietin (Epo) synthesized by neurons and astrocytes modulates the respiratory control system in adult mice. However, the impact of Epo in the regulation of the neural control of breathing during the postnatal development remains unknown. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate whether Epo interacts with the brainstem centers associated with respiration during postnatal life. Our immunohistochemical analysis revealed that Epo and Epo receptor (EpoR) are expressed in the main central respiratory nuclei at 1, 4, 7, 10, 15 and 21 days of age. Furthermore, by using antibodies to choline acetyltranferase (ChAT), somatostatin (SST) and neurokinin receptor 1 (NK1), EpoR was localized to the pre‐Bötzinger complex nucleus at all studied postnatal days. Whole body phtethysmography technique was used to evaluate basal ventilation and ventilatory response to hypoxia at 10% and 6% O2. Compared to wild type animals, Tg21 mice showed improved hypoxic ventilation (both at 10% and 6% O2) at postnatal days 15 and 21, but not 7 and 10. These results are in line with data obtained by radioimmunoassay, showing that overexpression of Epo in transgenic mice brains dramatically increases after 10 days of postnatal life. These results imply that cerebral Epo plays a key‐regulating role in the neural control of breathing in a period in which the central respiratory network undergoes full development and maturation.Source of research support is Molly Towell Perinatal Research Foundation (MTPRF).

Reduced Cerebral Blood Flow but Elevated Cerebral Glucose Metabolic Rate in Erythropoietin Overexpressing Transgenic Mice with Excessive Erythrocytosis

To examine the impact of excessive erythrocytosis on local cerebral blood flow (CBF) and cerebral glucose metabolic rate (CMR(glc)), we made use of our constitutively erythropoietin (Epo)-overexpressing transgenic mouse line (tg-6) that reach a mean hematocrit of 0.87. Compared with wild-type (wt) control siblings, CBF decreased by 44% in tg-6 mice, while upon hemodilution (tg-6-HD) to a physiologic hematocrit (e.g., 0.44) tg-6-HD mice returned the CBF to wt levels. Cerebral blood flow was determined in another transgenic mouse line that overexpresses human Epo in the brain only (tg-21): CBF increased by 17% compared with wt controls. However, oxygen delivery was similar in all four mouse groups tested (wt, tg-6, tg-6-HD and tg-21). Mean CMR(glc) was higher in tg-6 (+72%), tg-6-HD mice (+43%) and tg-21 (+22%) than in wt mice. Local CMR(glc) was higher in all 40 brain regions in tg-6 but only in 15 and 8 regions in tg-6-HD and tg-21 mice. These results show that prolonged increases in hematocrit did not alter cerebral oxygen delivery at a decreased CBF and increased CMR(glc). Hemodilution suggests that high blood viscosity is a cause of the decrease in CBF and partly of the increase in CMR(glc). Cerebral glucose metabolic rate may also be increased by a direct effect of Epo in the brain (tg-21 mice).

Brain‐derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK‐1/‐2 and Akt pathways

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.

Erythropoietin protects from axotomy‐induced degeneration of retinal ganglion cells by activating ERK‐1/‐2

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective activity upon reduced oxygenation or ischemia of brain, retina, and spinal cord. To examine whether Epo has an impact on the retrograde degeneration of retinal ganglion cells (RGCs) following optic nerve transection in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo preferentially in neuronal cells without inducing polycythemia. We show that the tg21 retina expresses human Epo and that RGCs in this mouse line carry the Epo receptor. Upon axotomy, the RGCs of Epo transgenic tg21 mice were protected against degeneration, as compared with wild-type control animals. Western blot analysis revealed decreased phosphorylation levels of STAT-5 and reduced expression of Bcl-XL in RGCs of axotomized tg21 animals, suggesting that the corresponding pathways are not crucial for Epo's neuroprotective activity. Increased phosphorylation levels of ERK-1/-2 and Akt, as well as decreased caspase-3 activity, however, were observed in injured tg21 retinae. Injection of selective inhibitors of ERK-1/-2 (PD98059) or Akt (Wortmannin) pathways into the vitreous space revealed that transgenic Epo protected the RGCs by a pathway involving ERK-1/-2 but not Akt. In view that axotomy-induced degeneration of RGC occurs slowly, and considering the earlier data on the safety and efficacy of Epo in human stroke patients, we predict the clinical implementation of recombinant human Epo not only in patients with acute ischemic stroke, but also with more delayed degenerative neurological diseases.

Neuron-selective erythropoietin (Epo) overexpression protects against transient focal ischemia and retinal ganglion cell axotomy in mice - role of Akt, Erk-1/2 and Jnk signaling

Background: It has previously been demonstrated that erythropoietin (Epo) protects against focal brain ischemia. Thus, clinical trials in human stroke patients have recently been initiated, which are currently performed. Unfortunately, the underlying mechanisms of action of Epo in vivo are still poorly understood. In order to elucidate this issue, transgenic mice overexpressing Epo in the central nervous system but not the rest of the body have recently been produced (so-called tg21 mice). We here examined the effects of Epo overexpression on neuronal injury after transient focal ischemia and retinal ganglion cell (RGC) axotomy.

Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin.

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

Retrograde Transport of Intact Poliovirus Through the Axon via the Fast Transport System

Intramuscularly inoculated poliovirus is thought to spread to the central nervous system through neural pathways in humans, monkeys, and the transgenic (Tg) mice carrying the human poliovirus receptor (PVR) gene. To gain insight into molecular mechanisms for the retrograde axonal transport of poliovirus, resulting in the expression of neurovirulence, a poliovirus-sensitive ICR-PVRTg21 mouse line (Tg21) was used as an animal model for poliomyelitis. We detected poliovirus antigens in axons of the sciatic nerve. All of the Tg21 mice, which had been inoculated into the calves with 1 × 106pfu of the Mahoney strain of type 1 poliovirus, showed symptoms of paralysis in the inoculated limbs (initial paralysis) within 48 h after the inoculation. The appearance of this initial paralysis was observed in mice whose sciatic nerves were transected at various times after virus inoculation. The results were indicators of the velocity of poliovirus transportation through the sciatic nerves under analysis. Poliovirus-related materials recovered from the sciatic nerve were mainly composed of intact 160S virion particles. The amount of 160S particle recovered was greatly reduced by coinjection with anti-PVR monoclonal antibody. These results suggest that one of the fast retrograde axonal transport systems is involved in poliovirus dissemination through the sciatic nerve and that IM-inoculated poliovirus is incorporated into the sciatic nerve as intact particles in a PVR-dependent manner, as it is in humans.

A Poliovirus-susceptible Transgenic Mouse Model as a Possible Replacement for the Monkey NeurovirulenceTest of Oral Poliovirus Vaccine

Abstract.Two poliovirus-susceptible transgenic mouse (Tg PVR) strains, Tg1 and Tg21, were compared with the monkey test for their sensitivity to neurovirulence of live oral poliovirus vaccine (OPV). Intracerebral (i.c.) and intraspinal (i.s.) routes of inoculation were investigated to determine the most suitable combination of mouse strain and route. Evaluation of the mouse tests was performed using several indicators; clinical score and failure time were selected as the most efficient. Tg1 and Tg21 mice inoculated i.s. with type 2, and Tg21 mice inoculated i.s. with type 3 OPV were determined to be the most appropriate systems, whereas they are shown not to be suitable for type 1 OPV. The sensitivity of each of the two mouse models was at least equal to that of the monkey test, suggesting that these mouse systems might be considered as a potential replacement for the monkey test of OPV. However, more data are needed to establish regulatory criteria of acceptability for vaccine lots tested in Tg PVR mice. This is the first study conducted with Tg PVR mice with all three types of poliovirus vaccine preparations.