Innervated adrenomedullary microphysiological system to model prenatal nicotine and opioid exposure
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Summary: All three reviewers were not convinced that this screening platform has been properly validated vis-à-vis the neurobiology of the adrenal gland, nor that it has a physiologic relevance for the understanding of living processes and organs.
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Abstract
The transition to extrauterine life results in a critical surge of catecholamines necessary for increased cardiovascular, respiratory, and metabolic activity. The mechanisms mediating adrenomedullary catecholamine release are poorly understood, given the sympathetic adrenomedullary control systems’ functional immaturity. Important mechanistic insight is provided by newborns delivered by cesarean section or subjected to prenatal nicotine or opioid exposure, demonstrating the impaired release of adrenomedullary catecholamines. To investigate mechanisms regulating adrenomedullary innervation, we developed compartmentalized 3D microphysiological systems (MPS) by exploiting the meniscus pinning effect via GelPins, capillary pressure barriers between cell-laden hydrogels. The MPS comprises discrete 3D cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, we demonstrate that adrenal chromaffin innervation plays a critical role in hypoxia-medicated catecholamine release. Furthermore, opioids and nicotine were shown to affect adrenal chromaffin cell response to a reduced oxygen environment, but neurogenic control mechanisms remained intact. GelPin containing MPS represent an inexpensive and highly adaptable approach to study innervated organ systems and improve drug screening platforms by providing innervated microenvironments.
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Reviewer #3:
In this manuscript, Soucy et al. describe a new technique that involves a 3D co-culture system that allows the analysis of the regulation of the sympathetic adrenomedullary system. The data demonstrate the advantage of such compartmentalized 3D systems relative to the 2 D system for long-term studies. The findings also show the usefulness of this system to understand the control by preganglionic sympathetic neurons of catecholamines released by the adrenal gland cells.
The main concern with the work relates to the uncertain physiological relevance of the co-culture system developed by the authors. Although I appreciate the utility of such reductionist techniques to understand how preganglionic sympathetic neurons regulate catecholamines released by the adrenal gland cells, this is too removed from a physiological setting.
It…
Reviewer #3:
In this manuscript, Soucy et al. describe a new technique that involves a 3D co-culture system that allows the analysis of the regulation of the sympathetic adrenomedullary system. The data demonstrate the advantage of such compartmentalized 3D systems relative to the 2 D system for long-term studies. The findings also show the usefulness of this system to understand the control by preganglionic sympathetic neurons of catecholamines released by the adrenal gland cells.
The main concern with the work relates to the uncertain physiological relevance of the co-culture system developed by the authors. Although I appreciate the utility of such reductionist techniques to understand how preganglionic sympathetic neurons regulate catecholamines released by the adrenal gland cells, this is too removed from a physiological setting.
It is difficult to judge the level of novelty of the MPS technique reported in this manuscript relative to what is in the previous paper (Ref 36) which is not available.
The innervation of tissues including heart and adrenal gland is highly specific. In addition to the circulating catecholamines secreted by the adrenal glands, cardiomyocytes are tightly controlled by direct innervation. Thus, whether co-culturing PNS with other cells mimic what happens in vivo is not clear.
The number of AMMCs displayed in figure 2B seems minimal as only very few cells were stained with cardiomyocyte markers. It would be interesting to know how many of these AMMCs receive innervation (Fig. 3E).
It is not clear how primary cardiomyocytes were exposed to the catecholamines emanating from the AMMCs? Were these co-cultured or were the cardiomyocytes exposed to the media of AMMCs?
Do the "n" in each figure represent cells or experiments (repeats)?
There is no description of the method used to quantify the immunofluorescent signal.
The Introduction is too long. It can easily be shortened to focus on the literature related to the topic.
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Reviewer #2:
Soucy and colleagues developed a thermoplastic microphysiological system (MPS) to investigate the mechanisms regulating adrenomedullary innervation. This system consists of 3D cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, they report that adrenal chromaffin innervation is critical for hypoxia-induced catecholamine release. They also show that opioids and nicotine affect adrenal chromaffin cell response to hypoxia without impairing neurogenic control mechanisms. In addition to providing mechanistic insights on adrenomedullary catecholamine release, this study represents an elegant proof-of-concept that the MPS have the potential to become useful tools to study organ innervation.
Disclosure: I do not have the expertise to review the …
Reviewer #2:
Soucy and colleagues developed a thermoplastic microphysiological system (MPS) to investigate the mechanisms regulating adrenomedullary innervation. This system consists of 3D cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, they report that adrenal chromaffin innervation is critical for hypoxia-induced catecholamine release. They also show that opioids and nicotine affect adrenal chromaffin cell response to hypoxia without impairing neurogenic control mechanisms. In addition to providing mechanistic insights on adrenomedullary catecholamine release, this study represents an elegant proof-of-concept that the MPS have the potential to become useful tools to study organ innervation.
Disclosure: I do not have the expertise to review the engineering aspects of this manuscript and will therefore share some concerns I have regarding the accuracy of this technology to mimic native tissues.
I understand that one advantage of the MPS over microfluidic devices using micro-posts or micro-tunnels is the presence of an unobstructed interface between the compartments that is similar to tissue interfaces. However, how better is it compared to other organs-on-chips constructs for reaching the biological complexity of an intact organ?
The system consists of adrenal chromaffin cells and preganglionic sympathetic neurons. I wonder if in this format it could lack the normal cellular heterogeneity of the adrenals. Can the absence of adrenal cortex cells producing aldosterone, androgens and glucocorticoids with important autocrine functions on chromaffin cells interfere with the ability of chromaffin cells to respond normally to a stimulus? Authors discuss that future efforts will incorporate additional adrenal cortical cell populations to better mimic the native physiology. Could they extend this discussion by highlighting the potential weaknesses of the model in its current format? Was any observations made that would suggest caveats?
In vivo, do all fibers innervating the medulla target the chromaffin cells or do some/most innervate the blood vessels or pericytes? If a majority of the innervation is to blood vessels, how does this system take into account potential changes in blood flow and perfusion of the adrenals that could occur and affect the oxygenation?
Early work suggests that adrenergic terminals innervate chromaffin cells and that the adrenal medulla receives a sympathetic and parasympathetic efferent and an afferent innervation (J Anat. 1993 Oct; 183: 265-276). How would this system allow to study such complex innervation? Is it possible to add additional neuronal types to this MPS?
In addition to the nicotinic cholinergic receptors, chromaffin cells express muscarinic receptors that may also be involved in catecholamine release. A quick profiling and comparison of the expression of the different receptors could reinforce the representative nature of the technology to model a biological system.
One important caveat of MPS is the challenge of delivering a drug in a physiologically realistic manner. Could the author comment on the doses of the different drugs used and how they are representative of what a chromaffin cell would normally "see" in vivo?
Could the authors comment on the culture media/conditions and how they are representative of a biological system? Would the use of blood or blood components be a better alternative to the system?
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Reviewer #1:
In this manuscript, Soucy and colleagues present a novel innervated system which they use to model the effects of prenatal nicotine and opioid exposure. Using the system they provide potentially interesting insights on how prenatal nicotine and opioid exposure could impact release of catecholamines. However, following careful review of the manuscript,I recommend that the authors provide substantial additional data and evidence to support the biological relevance of their findings.
Major points:
A main pillar of this manuscript is the assumption that the adrenal medulla is innervated. To substantiate their claims the authors cite books/book chapters, rather than citing convincing primary evidence. In fact, other than old EM images showing vesicular densities akin to synapses, I have not found published images of convincing …
Reviewer #1:
In this manuscript, Soucy and colleagues present a novel innervated system which they use to model the effects of prenatal nicotine and opioid exposure. Using the system they provide potentially interesting insights on how prenatal nicotine and opioid exposure could impact release of catecholamines. However, following careful review of the manuscript,I recommend that the authors provide substantial additional data and evidence to support the biological relevance of their findings.
Major points:
A main pillar of this manuscript is the assumption that the adrenal medulla is innervated. To substantiate their claims the authors cite books/book chapters, rather than citing convincing primary evidence. In fact, other than old EM images showing vesicular densities akin to synapses, I have not found published images of convincing axonal arborization in the adrenal medulla - if such images exist the authors should at least try to reproduce them for internal consistency of their study. This is particularly relevant if they wish to draw parallels between in vitro and in vivo systems. As this is a major pillar upon which this research stands, the lack of supporting histological evidence, which could be easily done, undermines the validity of this manuscript. Presenting primary evidence (i.e. not a textbook diagram) is essentia.
Multiple experiments lack appropriate controls. See comments on Figure 2B, 2D, Supplementary Figure 2.
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Summary: All three reviewers were not convinced that this screening platform has been properly validated vis-à-vis the neurobiology of the adrenal gland, nor that it has a physiologic relevance for the understanding of living processes and organs.
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