The multiple comparisons test analysis. This study

The Wilson’s lab
demonstrated the presence of SOS in an anesthetized, spinalized, carotid body
denervated in vivo rat preparation, proving that the SOS are active in
the normal physiological range and are stimulated strongly by hypoxia. To prove
oxygen sensing is occurring within the spinal cord (rather than brainstem or
other unidentified organ), The Wilson’s lab developed a novel adult rat,
artificially-perfused, thoracic spinal cord preparation. This preparation
consists of ribcage, spinal column, and descending aorta, but lungs, heart,
arterial chemoreceptors, brainstem and lumbar spinal cord were removed. This
preparation allows sympathetic activity to be recorded from the splanchnic
(sympathetic) nerve without compromising spinal cord perfusion. They have
identified that the spinal cord itself is capable of sensing oxygen, most
likely, the IML neurons. These previous experiments gave rise to my research
proposal.

Recently, I have identified
the SOS neurons through c-Fos immunohistochemistry after using the thoracic
spinal cord perfusion preparation, without nerve recording (Figure 1). The
preparation was perfused with an oxygenated aCSF combined with 1uM of TTX to
block synaptic transmission. TTX occludes the entrance of NaV1.4 sodium channel
(necessary for action potential discharge) by forming a network of hydrogen-bonds
at the outer lumen of the channelRJAW1 , therefore blocking
synaptic transmission (Chen & Chung,
2014). C-fos production was
triggered by either hypoxia or pressure challenges and compared to the
labelling on the Control group (Figure 2). The cell count revealed a
significant difference between the experimental groups and the Control group,
but no difference between experimental groups (Figure 3). Results were obtained
after ANOVA and Sidak’s multiple comparisons test analysis. This study revealed
the origin of the hypoxia and pressure responses seen on previous experiments.
Furthermore, I also performed a double-labelling
immunohistochemistry for the FOS protein combined with Choline Acetyl transferase
(ChAT) in order to explicitly show that the FOS activated neurons are localized
in the IML region. The methods used to perform the experiments mentioned above
are described in Aaim 1. The
expression of c-Fos protein has often been used as a marker of neural activity (Bullitt, 1990; Su,
Ho, Kuo, Wen, & Chai, 2009) and the combination with ChAT allows a better
localization of the IML neurons and discrimination between experimental groups.

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The expression
of  c-fos-like
protein may provide this kind

of  marker.

C-fos

is a proto-oncogene
that is expressed within

neurons following voltage-gated calcium entry into the cell

(Morgan and
Curran,

’86).

Neuronal  excitation 
leads to a

rapid and transient induction
of

c-fos

(Morgan et al., ’87).

 

C-fos

is a proto-oncogene
that is expressed within

neurons following voltage-gated C-fos is a proto-oncogene that is expressed within
neurons following voltage-gated calcium entry into the cell (James I. Morgan &
Curran, 1986). Neuronal excitation  leads to a rapid and transient induction of
c-fos (J. I. Morgan, Cohen,
Hempstead, & Curran, 1987). The protein 
product  can  be 
detected within  neurons  by immunohistochemical techniques 20-90  minutes after neu- ronal excitation, and
disappears 4-16 hours later (J. I. Morgan et al.,
1987).  Once expressed, c-fos protein enters the cell
nucleus and partici- pates in protein complexes that interact with DNA(Sambucetti &
Curran, 1986). These experiments gave foundation for posterior
experiments to be performed as of the neuronal identity and localization of the
SOS contributing to the legitimacy of the project.

Treatment with NaCN renders
cells hypoxic, resulting in a low oxygen tension condition similar to that
observed following ischemia and haemorrhage RJAW2 (Hill, Adrain, &
Martin, 2003; Kiang & Smallridge, 1994). Therefore, NaCN mimics a cytotoxic acute hypoxia
due to the blockage of oxidative phosphorylation CN? which is known to
increase intracellular free calcium concentration and reduce cell survival. For
this reason the SOS activity was measured through the splanchnic nerve in the
spinal cord preparation (Figure 4) under 500uM of NaCN (Figure 5). The SOS
activity to NaCN was then compared to the response to hypoxia (100 Torr O2/ 40
Torr CO2) given before and after the NaCN. SOS activity was triggered by NaCN,
which indicates that one of the mechanisms of responsible for the hypoxic
response might be mitochondrial.

As the spinal cord is part
of the CNS, it would be expected that the SOS are also sensitive to CO2 such as
the brain stem. However, not even severe hypercapnia (60 Torr of CO2, balanced
O2) was able to trigger the SOS response (figure 6) as even the mild hypoxia
does.

The main oxygen sensor, the
carotid body, is only activated after a certain period throughout mammal development.
Therefore, the question of, when is the onset of the SOS, was raised. So far,
the SOS was detected to be active on a 2 days old animal (Figure 7). As the SOS
are believed to have lifesaving capacities, it is very possible that the SOS
are active upon birth, however, due to the challenging technique, the P0 and P1
did not yield any results yet.

Being such an important component of the
respiratory network by promoting sympathetic responses under hypoxic
conditions, the SOS should also be present in other species, such as mice. The
presence of the SOS in mice adds the advantage of the genetic manipulation for further
KO studies. The SOS presence and sensitivity was then tested on mice and
detected since the first challenge of 400torr of PO2 (Figure 8).

Although the AMPK enzyme is
activated in response to a variety of metabolic stresses, such as hypoxia, it
does not seem to have any influence on SOS oxygen sensing. AMPK-KO mice were dissected
and reduced to a spinal cord preparation (see methods in aim 2) subjected to a
dose response study where the SOS response to decreasing levels of hypoxia was
test. The comparison between Control and KO groups revealed no difference
(Figure 9). Therefore, AMPK does not play any role on the SOS oxygen
sensitivity mechanism.

In order to demonstrate the functional significance
of the SOS in the respiratory network I employed the triple perfusion
preparation (methods described in aim 3 – Figure 10). On this preparation, when
the spinal cord is deprived of aCSF flow (therefore of oxygen as well) while
all other chemoreceptors (brainstem and carotid body) remain homeostatic, there
is a big increase in sympathetic activity (splanchnic nerve), a slight increase
in in vagus (brainstem) baseline activity as well as a subtle increase in ventilation
frequency. This demonstrates the retrograde carriage of information from the
spinal cord to the respiratory centre. On the other hand, when the flow is
stopped to the brain stem, there is a massive sympathetic activity increase,
followed as well as the presence of gasps a few seconds later in synchrony with
peaks of vagal activity. However, when the flow to the CSN is stopped (spinal
cord and brainstem combined) the amount and amplitude of gasps increases.
Interestingly, splanchnic activity seems to precede the gasps, while vagus
activity is synchronous with it (Figure 11).

 

SIGNIFICANCE

The sympathetic activation
derived from the SOS, resulting from a slight fall in CNS oxygenation (or
perfusion) shows how sensitive and therefore how important is the role of the
SOS on cardiorespiratory control. The proceedings of this research will require
the respiratory field to rethink sympathetic cardiovascular regulation and
adaptive responses to hypoxia.

These results introduce an
important new chapter in contemporary cardiorespiratory research. First, the
direct linkage between oxygen sensing and sympathetic activity has profound
consequences for our understanding of hypertension. Chronic conditions that
involve mild circulatory impairment and/or sustained hypoxia (obesity,
atherosclerosis, diabetes, asthma and COPD) may activate SOS to cause sustained
sympathetic drive, increased blood pressure, and hypertension. Second, in sleep
apnea, intermittent hypoxia leads to sustained increases in sympathetic and
respiratory activity, which is also associated with hypertension. Third, severe
hypotension caused by hemorrhage is met with profound sympathetic activity and
peripheral vasoconstriction. As severe hypotension is expected to reduce flow
to (and therefore oxygenation of) the CNS, the SOS mechanism is likely
activated. Fourth, reports that ischemia, a pathophysiological condition
associated with spinal cord stroke and injury, can activate the thoracic
sympathetic circuit may be explained by the SOS. Fifth, SIDS occurs in
vulnerable infants, unable to arouse and/or respond appropriately to an acute
cardiorespiratory crisis. The best hope for preventing SIDS is to discover why
some infants are unable to respond to cardiorespiratory crisis; then use this
knowledge to develop early-life screens so we can direct resources to infants
at greatest risk. The SOS appears to be a central player in physiological responses
to life-threatening cardiorespiratory events.

Indeed, oxygen sensitive preganglionic neurons are
well suited to the task of ensuring availability of oxygen in the CNS is
maintained: they are highly sensitive, the most direct and least
synaptic-dependent of any central sympathetic relay, and innervate practically
every end organ and blood vessel in the body. These properties of the SOS may
also serve as the trigger for survival mechanisms in response to acute hypoxia
during sudden respiratory and/or cardiac arrest, when brainstem-mediated
responses to carotid body oxygen sensors fail.

 RJAW1Lifted from: https://www.ncbi.nlm.nih.gov/m/pubmed/24607901/

 

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 RJAW2This is lifted from here:

 

https://link.springer.com/article/10.1023/A%3A1027363900317