Juan-Carlos Toledo-Salas, Ph.D.
Working Heart Brainstem Preparation
In this preparation a small (60g) rat is anesthetized with halothane, the lower body (legs, digestive, renal, reproductive systems) and skin are removed in a cold solution with Ringer solution. The brain cortex is removed.
Then it is put in a stereotaxic equipment and Ringer solution with 95%oxygen, 5%carbon dioxide is pumped into the aorta to replace blood.
The Working Heart Brainstem Preparation allows the recording of various nerves related to cardiorespiration functions, like the phrenic nerve, the abdominal nerve and the sympathetic nerve.
It also allows brain access for stimulating or recording neurons. Since thorax is exposed and most lungs removed it is not similar to in "in vivo" animals, but it provides tools for studying respiratory nerves and the brain respiratory system.
Phrenic nerve, abdominal nerve and sympathetic nerve during hypercapnia
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Effects of 10% hypercapnia exposure on phrenic, sympathetic and abdominal nerve activities.
The first trace shows the perfusion pressure (PP), the second trace the integrated sympathetic nerve (∫SN) activity, the third trace the sympathetic nerve (SN) activity, the fourth trace the integrated abdominal nerve (∫AN) activity, the fifth trace the abdominal nerve (AN) activity, the sixth trace the phrenic nerve (∫PN) activity, and the last trace the phrenic nerve (PN) activity. Lower records show the responses before and during hypercapnia
Working Heart Brainstem Preparation
WHBP
The purpose of my research was to evaluate the function of the retrotrapezoid nucleus (RTN) in the response to hypercapnia by evaluating its effects on respiratory nerves (phrenic, abdominal sympathetic) so RTN was lesioned with saporin before the WHFP experiments. In the WHBP the nerves during baseline and hypercapnia were registered but after making lesions in some rats and making a report I found that this research had been published 4 years earlier by Abdala et al, J Physiol. 2009 Jul 15; 587(Pt 14): 3539–3559 using the same methods and hypothesis. The only difference is that they decided to inhibit instead of lesioning the RTN, so baselines and hypercapnia recordings could be done in the same experiment while the RTN was active and inhibited.
In my experiments the phrenic nerve AUC sometimes changed to a square after lesioning the RTN, as in the paper published, but there were no histology equipments to verify where the lesion was done, so I made other type of analysis, as the response to hypercapnia was not homogenous and found there was a relation between the rhythm of phrenic nerve and breathing during baseline and their response to hypercapnia.
But...
They look similar ...
A better analysis was done using Poincare and entropy to relate baseline Phrenic nerve intervals (PNi) and Phrenic nerve amplitude (PNa) response to hypercapnia response. These results and a similar analysis to breathing amplitude (Ba) and breathing intervals (Bi) changes to hypercapnia in plesthysmography , using lesioned animals to get a larger range of response, were joined to make a manuscript about it, but unfortunately no histochemistry sections were done ( the brains I took were not kept properly) so the exact position of the lesion could not be determined.
To make a Poincare study, the phrenic nerve intervals where calculated before and during hypercapnia. The duration of one interval ( time between 2 peaks) is compared with the duration of the next interval. These are plotted and the SD1 (red line) and SD2 (green line) values are calculated as shown below. The proportion of SD1/SD2 during baseline was compared to the amplitude increase during hypercapnia.
Here it is shown during baseline
and hypercapnia the Poincare plots Phrenic nerve intervals (PNi) during low <105, medium 105-125, and high >125 phrenic nerve amplitude responses to hypercapnia.
There was not a clear relation when considering the area under the curve instead of the maximum activity. There was just a weaker approximate entropy relationship. This could be because the intervals between maximum phrenic nerve activity reflect maximum neuronal activity and the phrenic nerve area under the curve considers non maximum phrenic nerve activity.
A similar test was done by measuring the breathing intervals in plethysmography, but here a more clear relationship was found when calculating the sample entropy.
The same analysis was done but measuring changes in baseline phrenic nerve amplitudes before and after hypercapnia. These are explained in a manuscript of the Papers section of the webpage.