H5N1 infection transiently reduces the number of TH-positive DA neurons in the SNpc
Infection with H5N1 leads to a wide array of pathologies in humans and mice, including diffuse damage to the lung, hemophagocytic damage to spleen, lymph nodes and blood vessels and alterations in bone marrow (Dybing et al., 2000
; Nishimura et al., 2000
; Yuen and Wong, 2005
; Korteweg and Gu, 2008
). Additionally, several strains of H5N1 have been identified that are neurotrophic in mice, inducing inflammation, neuronal death and induction of pathologies seen in Parkinson’s disease, including formation of aggregates of phosphorylated alpha-synuclein (Wang et al., 2008
; Jang et al., 2009
To determine if H5N1 infection can directly induce the parkinsonism pathologies by damaging dopaminergic neurons, we stereologically assessed the number of tyrosine hydroxylase positive (TH+) DAergic neurons in the SNpc () and empirically determined the total amount of DA, HVA, and DOPAC in the SN, brainstem (), cortex and hippocampus (data not shown).
H5N1 infection alters TH-positive neurons in SNpc
Percent Change in the DA, DOPAC, and HVA in striatum and brainstem following systemic H5N1 Infection
At day 10 dpi, we find an approximately 60% loss of TH-positive neurons in the SNpc, compared to non-infected control mice. By 60 dpi, we saw a recovery in the TH+ DAergic neuron number and by 90 dpi, we saw no difference in the number of SNpc TH+ DA neurons (). From 10 through 60 dpi, the TH+ DAergic neurons appeared shrunken and atrophic. The longest length of TH-positive neuronal cell bodies was reduced by 20%, however the size of these cells recovered and appeared similar to that seen in the non-infected control mice at day 90 dpi ().
To determine if the loss of TH-positive neurons was a result of cell death and the subsequent recovery in number was due to repopulation via neurogenesis we examined expression of activated caspase-3, TUNEL and FluoroJadeB staining as well as expression of Ki-67. In regions of the brain where A/VN/1203/04 virus had been detected, we found a few activated caspase-3-positive apoptotic but not FluoroJadeB necrotic cells (data not shown). The apoptotic cells were only visible through 10dpi, and although not systematically quantitated, did not appear to be numerous enough to account for a 20% reduction in SN TH-positive number. Examination of cells in the SNpc showed no evidence of cell division using immunohistochemical (Ki-67) methods through 90 dpi, and therefore there is no compelling evidence that SNpc DA neurons underwent any form of neurogenesis. Thus, it is most likely that active H5N1 infection induces SNpc DAergic neurons to transiently reduce their metabolic capacity that both compromises TH activity and reduces cell size (), each leading to a loss of the DAergic phenotype in neurons.
Effect of H5N1 infection on Dopamine, DOPAC and HVA levels in the CNS
We used reverse phase HPLC with electrochemical detection to determine if infection with H5N1 affected the levels of dopamine and its metabolites in the striatum, which is the major target of SNpc DAergic neurons, brainstem, substantia nigra and cortex. The amount of striatal DA and its metabolites, DOPAC and HVA, were each significantly decreased by approximately 40% compared to intranasal saline treated control mice at 10 dpi. By 60 dpi, DA levels returned to baseline levels and this change was stable through 90 dpi. This pattern of a transient decrease at 10 dpi followed by recovery at 90 dpi was also seen when examining levels of HVA and DOPAC ().
We also compared the turnover ratio of DA in the striatum ((DOPAC + HVA)/DA) to see if infection with H5N1 altered DA metabolism. Despite alterations in levels of DA, its turnover was unchanged due to concurrent fold-changes in DOPAC and HVA.
In brainstem, the pattern of DA levels was reversed. At 10 dpi, we found a transient 300% increase in DA and DOPAC and a 500% increase in HVA () that resolved by 60 dpi. Like, striatum, no changes in DA turnover were detected since the relative ratios of DA to DOPAC and HVA did not change at each timepoint.
No changes in DA, DOPAC or HVA levels were detected in substantia nigra or cortex at any time after influenza infection (data not shown).
Effect of H5N1 infection on NE and 5-HT levels in the CNS
We used reverse phase HPLC with electrochemical detection to determine if infection with H5N1 affected levels of NE, 5-HT and its metabolite, 5-HIAA in the SN, striatum, brainstem, cortex and the hippocampus.
In striatum, we observed a slight increase in striatal NE at 10 dpi that resolved to baseline levels by 60 dpi (). Unlike NE, 5-HT levels dropped by approximately 60% and remained low through 90 dpi. A similar reduction in 5-HIAA was detected, although there was a slight recovery by 90 dpi to 60 percent of baseline ().
Percent Change in the Amount of NE in Brain Following H5N1 Infection
Percent Change in 5-HT and 5-HIAA in Brain Following H5N1 Infection
In SN, we measured a slight increase in NE at 10 dpi, although this change did not reach statistical significance (). In regard to 5-HT, we observed a 50% decrease that stayed significantly below control levels through 90 dpi (). A similar reduction was observed in 5-HIAA levels, although by 90 dpi, these levels returned values significantly similar to control animals ().
In hippocampus, no changes in NE levels were seen until 60 dpi, where we observed a 75% increase in NE () that returned to baseline levels at 90 dpi. No statistically significant changes were seen in 5-HT or 5-HIAA levels following H5N1 infection compared to control mice ().
In cortex, we measured a significant 70% reduction in NE at 10 dpi, that recovered to control levels by 60 dpi (). In regard to 5-HT, we observed a 90% decrease that stayed significantly below control levels through 90 dpi. A similar reduction was observed in 5-HIAA levels ().
No changes in NE, 5-HT or 5-HIAA were detected in brainstem.
H5N1 infection increases the number of microglia in the SNpc
Microglia are the resident immune cells of the CNS, derived from cells in the monocyte lineage (Hickey and Kimura, 1988
; Simard and Rivest, 2004
). When in their surveillance mode, they are said to be resting (Gehrmann et al., 1993
; Raivich, 2005
) and have a characteristic histological appearance with long slender tendrils emanating from their cell body (). Once exposed to infection, injury or trauma (Harry and Kraft, 2008
) they undergo a transformation in which they retract and thicken their processes and assume a more ameboid morphology () (Graeber and Streit, 2010
). To determine if exposure to H5N1 alters the morphology and number of microglia in the SNpc, we used the optical fractionator to assess the number of resting and activated microglia. Control C57BL/6 mice administered saline intranasally were found to have approximately 7500 total Iba-1-positive microglia in the SNpc. Of these, approximately 10% of these were structurally classified as activated, while 90% were classified as resting. Sixty days after intranasal innoculation of H5N1, we found a 67% increase in total microglial number. Examination of microglial subtype revealed a 300% increase in activated microglia and a 33% increase in resting microglia. The increase in total microglia number, as well as percent increase in activated and resting microglia, were unchanged from day 60 dpi to day 90 dpi (). This suggests that neurotropic influenza exposure in the brain induces a long-term, if not permanent, increase in activated microglia.
H5N1 Infection Increases the Number of Activated Microglia in the SNpc
Effect of H5N1 infection on levels of cytokines and chemokines in the lung and CNS
Activated microglia have been shown to produce a variety of cytokines, chemokines, and growth factors following exposure to infection as well as other insults to the CNS (Hirsch et al., 2003
; Kim and Joh, 2006
; Tansey and Goldberg, 2010
). This “inflammatory response” has been shown to be varied and specific to the type of insult (Perry et al., 2003
). Some cytokines (IL-1α, IL-1β, IL-2, IL-9, IL-12, IFNγ and TNFα) function primarily to induce inflammation (pro-inflammatory), while others (IL-6, IL-10, and IL-13) suppress inflammation (Opal and DePalo, 2000
). A class of cytokines function as chemokines, acting as chemoattractants and include eotaxin, KC, IP-10, MCP-1, MIP1α, and MIP1β (Fernandez and Lolis, 2002
), while others can act as growth and differentiation factors (GM-CSF, M-CSF and VEGF) (Metcalf, 1985
). In this study, we examined cytokine, chemokine and growth factor profiles in regions of the CNS that were infected by H5N1 infection, both during (day 0–21) and after (day 60–90) the acute infectious stage (Jang et al., 2009
). To determine if any alterations were specific to the CNS or were in response to, or coincident with, humoral activation of cytokines, we also measured these proteins in lung, which is the primary site of H5N1 influenza infection in mice (Jang et al., 2009
) and traditionally, in humans (Yuen and Wong, 2005
Examination of cytokines expression profiles in tissues generally demonstrated 4 distinct profiles of induction: In the first pattern, proteins were transiently increased during the initial phase of infection through day 10 dpi and then returned to baseline levels. A second pattern of induction showed an initial decrease in expression followed by a continued loss or a return to baseline levels. A third pattern of cytokine/chemokine expression demonstrated an initial transient increase in expression followed by a return to baseline levels and then a re-induction at times after the influenza virus was no longer detectable by immunohistochemical methods (visualization of NP protein). A fourth pattern of cytokine/chemokine expression was observed where there were no changes during the active phase of infection (through day 10 dpi), but at a later time, induction was detected. Examples of these patterns of expression are seen in .
Patterns of Cytokines, Chemokines and Growth Factors Expression Observed Following H5N1 Infection
In the lung, the expression of proinflammatory cytokines and chemokines, displayed all 3 of these distinct patterns, while 1 showed a unique pattern not seen in any of the brain regions, where there was a sustained decrease in expression. First, we saw that some of these proteins were transiently increased during the initial phase of infection (through day 21 dpi) and then returned or decreased to baseline levels. The pro-inflammatory cytokines/chemokines that expressed this profile were IL-6, IL-12, GM-CSF, G-CSF, IFNγ, IP-10, KC, MCP-1, MIP1β, MIP1α, and TNF
IL-10, an an
iinflammatorycytokine/chemokine also expressed this profile. A second pattern of induction showed an initial transient decrease in expression followed by a return to baseline levels. The pro-inflammatory cytokines/chemokines that expressed this profile were IL-1β IL-2, eotaxin and VEGF. The cytokine/chemokine pattern 4 expression was also demonstrated in lung where there is an initial transient increase in expression followed by a return to baseline levels, and then a reinduction at times after the influenza virus was no longer detectable by immunohistochemical methods (visualization of NP protein). The pro-inflammatory cytokines/chemokines that expressed this profile were IL-1α and M-CSF. The fourth pattern, where there is induction, return to baseline and then reinduction was seen in the response of IL-1α, eotaxin, and G-CSF. IL-9 had a unique response, where there was a down-regulation without return to baseline, while IL-13 was not detected in lung ().
Expression of Cytokines, Chemokines and Growth Factors in the Lung Following intranasal H5N1 Infection
In the CNS, we examined the expression of cytokines and chemokines in 4 separate regions: brainstem (), substantia nigra (), striatum () and cortex ().
Expression of Cytokines, Chemokines and Growth Factors in the Brainstem Following Intranasal H5N1 Infection
Expression of Cytokines, Chemokines and Growth Factors in the Substantia Nigra Following intranasal H5N1 Infection
Expression of Cytokines, Chemokines and Growth Factors in the Striatum Following intranasal H5N1 Infection
Expression of Cytokines, Chemokines and Growth Factors in the Cerebral Cortex Following intranasal H5N1 Infection
In the brainstem, the expression of cytokines, chemokines and growth factors displayed 2 of the different patterns described above. The pro-inflammatory cytokines, chemokines and growth factors that expressed the first profile were IL-1α, IL-12 (p70), IL-13, eotaxin, G-CSF, GM-CSF, IP-10, KC, M-CSF, MCP-1, MIP-1α, MIP-1β, and TNF-α. The anti-inflammatory cytokines IL-10 also followed this profile. The level of proinflammatory cytokines and growth factors IL-1β, IL-2, and VEGF displayed pattern 4. They were not changed immediately upon detection of the virus, but increased later when NP protein was no longer evident in the region. ().
In the substantia nigra, the expression of cytokines, chemokines and growth factors displayed profiles 1, 3, and 4, described above. The pro-inflammatory cytokines, chemokines and growth factors that expressed the first profile were IL-1β, IL-2, IL-6, G-CSF, M-CSF, and MCP-1 where their expression increased at 3 or 10 dpi and then returned to baseline levels. IL-13 exhibited the third pattern listed above, with its levels increasing prior to day 21 followed by a return to baseline and another rise in level at a later point after the active infection (60 dpi) was over. SN levels of the chemokines and growth factors GM-CSF and MIP-1β exhibited pattern 4, where expression did not change immediately upon detection of the virus, but increased later when NP protein was no longer evident in the region. Neither MIP-1α nor TNF-α was detected in the SN following exposure to influenza ().
In striatum, the expression of cytokines, chemokines and growth factors displayed profiles 1, 3, and 4, described above. The pro-inflammatory chemokines and growth factors that expressed profile 1 were eotaxin and M-CSF. The cytokine that expressed profile 3 was IL-2, while the anti-inflammatory IL-10 displayed profile 4. IL-1α, IL-6, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFN- γ, MIP-1α, MIP-1 β, and TNF-α were not detected in striatum following exposure to H5N1 influenza ().
In cortex, the expression of cytokines, chemokines and growth factors displayed profiles 1, 2 and 4. The pro-inflammatory cytokines that expressed profile 1 were IL-2 and IL-9. The growth factor VEGF expressed profile 2 while an anti-inflammatory cytokine, IL-10 displayed profile 4. IL-1α, IL-6, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFN-γ, MIP-1α, MIP-1 β, or TNF-α were not detected in cortex following exposure to influenza ().
Effect of dopamine on cytokine expression in SN cell culture
The reinduction of cytokine/chemokine expression between 60 and 90 days -long after the H5N1 virus was absent from the CNS- appeared to coincide temporally with the re-expression of tyrosine hydroxylase in the SN and appearance of DA in the striatum. To determine if some of the inflammatory response was induced by this reintroduction of dopamine to the basal ganglia we examined the pattern of cytokine expression using an in vitro postnatal substantia nigra culture system (Smeyne and Smeyne, 2002
). We allowed SN cultures to stabilize in a 2% O2
, 5% CO2
environment for 1 week and then added either 500 nM or 5 µM DA to each culture. Supernates were taken at 8 hours, 24 hours and 48 hours after the addition of dopamine and 22 cytokines/chemokines were measured. Of the 22 cytokine/chemokines measured, 12 were detected in the supernates (). The addition of dopamine increased expression of 4 of the 12 detected cytokine/chemokines including IL-1β (24 hours after addition of 5 µM DA), TNFα (8 hours after 500 nM DA), IL-13 (8 hours after 500 nM DA) and GM-CSF (24 hours after at 5 µm DA).
Expression of Cytokines, Chemokines and Growth Factors at 8, 24 and 48 hours following addition of dopamine to primary substantia nigra cultures