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. 2018 Nov 2;597(1):41–55. doi: 10.1113/JP277008

Baroreflex functionality in the eye of diffusion tensor imaging

Ching‐Yi Tsai 1, Yan‐Yuen Poon 1,2, Julie Y H Chan 1,✉, Samuel H H Chan 1,✉
PMCID: PMC6312450  PMID: 30325020

Abstract

By applying diffusion tensor imaging (DTI) as a physiological tool to evaluate changes in functional connectivity between key brainstem nuclei in the baroreflex neural circuits of mice and rats, recent work has revealed several hitherto unidentified phenomena regarding baroreflex functionality. (1) The presence of robust functional connectivity between nucleus tractus solitarii (NTS) and nucleus ambiguus (NA) or rostral ventrolateral medulla (RVLM) offers a holistic view on the moment‐to‐moment modus operandi of the cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone. (2) Under pathophysiological conditions (e.g. neurogenic hypertension), the disruption of functional connectivity between key nuclei in the baroreflex circuits is reversible. However, fatality ensues on progression from pathophysiological to pathological conditions (e.g. hepatic encephalopathy) when the functional connectivity between NTS and NA or RVLM is irreversibly severed. (3) The absence of functional connectivity between the NTS and caudal ventrolateral medulla (CVLM) necessitates partial rewiring of the classical neural circuit that includes CVLM as an inhibitory intermediate between the NTS and RVLM. (4) Sustained functional connectivity between the NTS and NA is responsible for the vital period between brain death and the inevitable cardiac death. (5) Reduced functional connectivity between the NTS and RVLM or NA points to inherent anomalous baroreflex functionality in floxed and Cre‐Lox mice. (6) Disrupted NTS‐NA functional connectivity in Flk‐1 (VEGFR2) deficient mice offers an explanation for the hypertensive side‐effect of anti‐vascular endothelial growth factor therapy (anti‐VEGF) therapy. These newly identified baroreflex functionalities revealed by DTI bear clinical and therapeutic implications.

Keywords: Arterial baroreflex, Diffusion tensor imaging, Neural circuits

Introduction

Whereas the literature abounds with studies using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) on forebrain structures, rarely is this approach applied to the brainstem, particularly in small animals. By successfully imaging changes in functional connectivity between key brainstem nuclei involved in the baroreflex circuits of mice and rats, recent work, primarily from our group (Tsai et al. 2013, 2015, 2017 a, b ; Su et al. 2016), has offered some new and controversial insights on DTI in general, and functionality of the baroreflex in particular. By showing that disrupted functional connectivity can be reversed, we demonstrated that DTI can be used as a physiological tool in addition to its traditional use as a morphological and pathological tool. The feasibility of visualizing the baroreflex neural circuits in the brainstem in conjunction with radiotelemetric evaluation of cardiovascular responses has unveiled several hitherto unidentified phenomena that will form the core of this Topical Review.

Classical neural circuits of the baroreflex

By providing a feedback mechanism that dampens fluctuations in cardiovascular parameters induced by perturbations inside or outside the body, baroreflex functions as the most fundamental mechanism in short‐term (Cowley et al. 1973) and long‐term (Thrasher, 2004) regulation of blood pressure (BP) and heart rate (HR). The classical neural circuits of the baroreflex were established based on results initially from anatomical (tracers), physiological (stimulation or lesion) and pharmacological (agonists and antagonists) experiments (Ross et al. 1985; Ruggiero et al. 1989; Dampney, 1994; Spyer, 1994) and expanded later to include Fos expression work (Chan & Sawchenko, 1998; Dampney & Horiuchi, 2003). According to this well‐established circuit (Fig. 1 A and B), an increase in impulse traffic in the primary baroreceptor afferents on detection of augmented BP by the arterial baroreceptors will lead to excitation of neurons in the nucleus tractus solitarii (NTS). From there, two physiological consequences will follow to normalize BP. In cardiac vagal baroreflex (Fig. 1 A), excitation of neurons in the nucleus ambiguus (NA) by fibres from the NTS reduces HR via the vagus nerve. Outputs from the NTS also elicit a baroreflex‐mediated decrease in sympathetic vasomotor tone (Fig. 1 B) by inhibiting the rostral ventrolateral medulla (RVLM), a site that exerts a tonic excitatory action on the sympathetic preganglionic neurons in the intermediolateral column (IML) of the thoracic spinal cord, via activation of the caudal ventrolateral medulla (CVLM). Conversely, a series of events in the opposite direction will take place to normalize a reduction in BP. Impairment of baroreflex functionality therefore results inevitably in a hypertensive or hypotensive state, and death under extreme conditions when the baroreflex is defunct (Chan et al. 2005 a).

Figure 1. Classical and revised baroreflex neural circuits.

Figure 1

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A and B, in the classical neural circuits, primary afferent inputs from the arterial baroreceptors activate neurons in the nucleus tractus solitarii (NTS). Outputs from the NTS elicit the cardiac vagal baroreflex (A) by exciting neurons in the nucleus ambiguus (NA) that in turn inhibits the heart via the vagus nerve. Outputs from the NTS also elicit the baroreflex‐mediated sympathetic vasomotor tone (B) by activating the caudal ventrolateral medulla (CVLM), which inhibits the rostral ventrolateral medulla (RVLM), a site that exerts a tonic excitatory action on the sympathetic preganglionic neurons in the intermediolateral column (IML) of the thoracic spinal cord. C and D, there are two rewiring options in the revised neural circuit for the baroreflex‐mediated sympathetic vasomotor tone to accomplish the mandatory inhibitory action of the NTS on the RVLM in the absence of CVLM. First, an excitatory projection from the NTS terminates on inhibitory interneurons that synapse on spinally projecting RVLM neurons (C). Second, an inhibitory projection from the NTS terminates directly on RVLM neurons that project to the IML in the spinal cord (D).

Diffusion tensor imaging opens a new frontier in the evaluation of baroreflex functionality

Until recently, whether neuronal traffic in the above‐mentioned neural circuits actually takes place in the brainstem during the execution of baroreflex has never been visualized. Based on successful implementation of tractographic analysis of the medulla oblongata in mice (Tsai et al. 2013, 2015, 2017 a; Su et al. 2016) and rats (Tsai et al. 2017 b), this visualization is now feasible. More importantly, the degree of prevalence of traffic within the neural circuits of cardiac vagal baroreflex and baroreflex‐mediated sympathetic vasomotor tone has been shown to bear physiological, pathophysiological and pathological implications.

Basic principles of DTI and tractographic analysis

DTI was introduced in the mid‐1990s (Basser et al. 1994) as a means to provide a new image contrast in MRI, namely, estimation of axonal organization in the living brain. Since DTI uses water motion as a probe to infer neuroanatomy, the basic principle that governs this technique is Brownian motion of molecules, in this case, water molecules, during diffusion. As illustrated in Fig. 2 A, in free diffusion, the probability distribution of water molecules in random movement is in the shape of a circle and is called isotropic diffusion. However, in restricted diffusion, the probability distribution of water molecules is in the shape of an ellipsoid and is called anisotropic diffusion, with the main or elongated axis (the tensor) oriented in parallel with the principal diffusion direction within a voxel. In the white matter (Fig. 2 B), the presence of insulating myelin sheath that wraps around the axon creates a boundary that dictates diffusion along the fibre bundles as the main direction of diffusion (axial diffusivity; λ∥), and the probability of water molecules crossing the axon (radial diffusivity; λ⊥) is low.

Figure 2. Principles of DTI as an experimental tool to measure functional connectivity.

Figure 2

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A, in free diffusion, the probability distribution of water molecules in random movement is in the shape of a circle and is called isotropic diffusion. However, in restricted diffusion, the probability distribution of water molecules is in the shape of an ellipsoid and is called anisotropic diffusion. B, in the white matter, the boundary created by the myelin sheath that surrounds the axon dictates that the main direction of diffusion is along the fibre bundles (axial diffusivity; λ∥), and the probability of water molecules crossing the axon (radial diffusivity; λ⊥) is low. Fractional anisotropy (FA), which ranges from 0 (maximum isotropy) to 1 (maximum anisotropy), is the most widely used index for DTI. FA is derived from the standard deviation of the lengths of the longest, middle and shortest axes (called eigenvalues, λ1, λ2, λ3) of the three diffusion ellipsoids created by water movement in three dimensions. C, since the passage of action potentials creates prominent anisotropy of water molecules along the long axis of the axon bundles, changes in FA can be taken to infer altered impulse traffic between two brain structures, which we termed functional connectivity.

The most widely used index for DTI is fractional anisotropy (FA), which represents the directional variation in the signals detected by the MR scanner that are related to those two forms of diffusivity. Mathematically, FA, which ranges from 0 (maximum isotropy) to 1 (maximum anisotropy), is derived from the standard deviation of the lengths of the longest, middle and shortest axes (called eigenvalues, λ1, λ2, λ3) of the three diffusion ellipsoids created by water movement in three dimensions. Tract reconstruction by linking pixels with similar orientation of the longest axis of the diffusion tensor, a method called tractography, provides information on the three‐dimensional orientation of fibres. Overall, axonal membranes are shown to be the primary determinant of diffusion anisotropy of water in neural fibres, while myelin can modulate the degree of anisotropy in a given tract. Tractographic signals therefore do not represent the true nerve fibres. Instead, by measuring the movement of all water molecules over a voxel, DTI provides an estimate of the principal direction of water movement along a group of axons in a pathway. Representative colours for tractography in colour‐encoded FA maps (Fig. 3) are commonly assigned to denote functional connectivity between two regions of interest, for example, blue to show caudal‐rostral; red to show left‐right and green to show dorsal‐ventral. Readers may refer to excellent reviews (Mori & van Zijl, 2002; Mori & Zhang, 2006; Alexander et al. 2007) or monograph (Johansen‐Berg & Behrens, 2013) for detailed information on DTI and tractography.

Figure 3. Experimental scheme to visualize baroreflex neural circuits at work.

Figure 3

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A typical DTI procedure involves (1) obtaining high quality sagittal anatomical reference imaging of the brain, followed by T2‐weighted (T2WI) coronal anatomical imaging of a restricted brainstem area that contains the medullary portion of NTS, NA, RVLM or CVLM, and (2) imaging the robust connectivity between these medullary nuclei using a spin‐echo‐planar imaging‐DTI sequence. (3) Finally, tractography, a method of tract reconstruction by linking pixels with similar orientations of the longest axis of the diffusion tensor, provides information on the three‐dimensional orientation of fibres. Representative colours for tractography in colour‐encoded FA maps are commonly assigned to denote connectivity between two regions of interest, for example, blue to show caudal‐rostral, red to show left‐right and green to show dorsal‐ventral. The DTI procedures, when performed at selected time points in parallel with temporal changes in blood pressure (BP), heart rate (HR) and indices of cardiac vagal baroreflex (BRS) or baroreflex‐mediated sympathetic vasomotor tone (BLF) obtained by radiotelemetry coupled with power spectral analysis, will reveal reversible or irreversible alterations of the baroreflex neural circuits that bear crucial clinical implications.

DTI as a tool for morphological or pathological analysis

Based on the observations that DTI of live and fixed brains provides similar results (Sun et al. 2003), the conventional wisdom is that this method provides information primarily on static anatomy and is less influenced by physiology (Mori & Zhang 2006). In terms of pathology, earlier animal work suggested that demyelination is associated with an increase in radial diffusivity (Song et al. 2005) and axonal injury is associated with a decrease in axial diffusivity (Budde et al. 2009), possibly because of disarray of the axons. It is therefore not surprising that neural fibre density determined by DTI is highly correlated with brain abnormalities associated with neural degeneration (Oishi et al. 2011; Ofori et al. 2015) and white matter disorders (Horsfield & Jones, 2002) such as multiple sclerosis (Audoin et al. 2007), ischaemic leukoariaosis (O'Sullivan et al. 2004), traumatic brain injury and cerebral stroke (Huisman, 2003). The variation in diffusion indices might be affected by multiple factors, including axonal loss or fibre degeneration, demyelination of the fibres, gliosis and loss of fibre integrity (Ellis et al. 1999; Abe et al. 2002; Sun et al. 2006; Lin et al. 2017).

DTI is also a tool for functional analysis

Recent observations from our group strongly support the notion that DTI can also be employed as an experimental tool for functional analysis. Our rationale is that since the passage of action potentials creates prominent anisotropy of water molecules along the long axis of the axon bundles (Fig. 2 C), an increase, decrease or disappearance of FA can be taken to infer augmentation, reduction or cessation of impulse traffic between two brain structures, which we termed elevation, disruption or severance of functional connectivity. As elaborated in detail in subsequent sections, we found that the changes in FA between key nuclei in the baroreflex neural circuits are commensurate with the manifested functionality of the reflex. More intriguing is the observation that the disrupted functional connectivity may be reversible. This suggests that we are dealing with a physiological rather than morphological or pathological phenomenon.

Visualizing baroreflex neural circuits at work in the brainstem is now feasible

Unlike in the forebrain, application of DTI to the brainstem and tractographic analysis of the baroreflex neural circuits, particularly in mice, require extraordinary efforts. In addition to the constraints of size, access to the brainstem is difficult because it is located beneath the cerebellum and in an oblique position from the horizontal plane of the cortical surface. We solved this problem by using a high‐performance transmitter‐receiver surface cryo‐probe coil to improve signal detection from the head of the mouse placed with its cortex in a horizontal position. We also implemented systematic optimization of the scanning parameters that allowed us to obtain high‐quality T2‐weighted sagittal anatomical reference imaging of the brain (Table 1) and coronal anatomical imaging of a restricted brainstem area that contains the medullary portion of NTS, NA, RVLM or CVLM (Table 2); and to image the robust functional connectivity between these medullary nuclei using a spin‐echo‐planar imaging‐DTI sequence (Table 3). The specificity of these findings is revealed when significant changes are always reported in terms of axial diffusivity (λ∥) rather than radial diffusivity (λ⊥). Furthermore, tractographic analysis of the bilateral pyramidal tracts, which do not play a role in cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone, showed consistent DTI images and FA values only in the caudal‐rostral direction (Su et al. 2016; Tsai et al. 2017 a). Most significantly, these tractographic data revealed reversible or irreversible alterations of the baroreflex neural circuits that bear crucial clinical implications when viewed in parallel with temporal changes in BP, HR and experimental indices of cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone obtained by radiotelemetry coupled with power spectral analysis (Fig. 3).

Table 1.

Optimal parameters for high resolution T2‐weighted sagittal anatomical reference imaging of the brain of the mouse, using multislice turbo rapid acquisition with refocusing echoes (Turbo‐RARE) sequence

Parameter
Field of view 18.0 mm × 15.0 mm
Matrix dimension 384 × 320 pixels
Spatial resolution 47 μm × 47 μm
Slice thickness 200 μm
Interslice distance 200 μm
Echo time 12.6 ms
Effective echo time 25.3 ms
Repetition time 3000 ms
RARE factor 4
Number of averages 3
Total acquisition time 12 min
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Table 2.

Optimal parameters for high resolution T2‐weighted coronal anatomical imaging of a restricted area of the brainstem that covered the medullary portion of the nucleus tractus solitarii (NTS), nucleus ambiguus (NA) and rostral and caudal ventrolateral medulla (RVLM, CVLM) of the mouse, using multislice turbo rapid acquisition with refocusing echoes (Turbo‐RARE) sequence

Parameter
Field of view 12.0 mm × 12.0 mm
Matrix dimension 192 × 192 pixels
Spatial resolution 62 μm × 62 μm
Slice thickness 200 μm
Interslice distance 200 μm
Echo time 12.5 ms
Effective echo time 25 ms
Repetition time 3000 ms
RARE factor 4
Number of averages 5
Total acquisition time 12 min
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Table 3.

Optimal parameters for diffusion tensor imaging (DTI) to determine the functional connectivity between the NTS and NA, RVLM of CVLM of the mouse, using spin echo‐planar imaging‐DTI sequence in the coronal plane covering the same ten 200‐μm slices in the T2‐weighted coronal reference images without gap

Parameter
Field of view 12.0 mm × 12.0 mm
Matrix dimension 128 × 128 pixels
Spatial resolution 94 μm × 94 μm
Slice thickness 200 μm
Interslice distance 200 μm
Echo time 24 ms
Repetition time 2500 ms
Number of diffusion directions 46
Optimized b value/direction 1500 s/mm2
Gradient duration 4.1 ms
Gradient separation 10.3 ms
Number of averages 15
Acquisition time 33 min 45 s
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Given the size of the NTS, NA and RVLM in mice, optimization of the scanning parameters has in fact been a daunting task. Among the multiple factors to be considered, the reciprocal contribution of signal‐to‐noise ratio (SNR) and resolution to image quality (Johansen‐Berg & Behrens, 2013) warrants further elaboration. Two factors relevant to SNR are voxel volume and number of acquisitions (averages). Voxel volume determines the number of water molecules that contribute to the signal to be detected. A voxel of 2 × 2 × 2 mm3 has an 8 times higher SNR than a 1 × 1 × 1 mm3 voxel. Averaging repeated acquisitions reduces noise by the square root of the number of averages. To increase SNR by a factor of 2 requires 4 averages. As illustrated above, to increase the spatial resolution from 2 × 2 × 2 mm3 to 1 × 1 × 1 mm3, the SNR is reduced by a factor of 8. To recover the original SNR would require 82 = 64 averages.

As indicated in Table 3, our observations were based on DTI sequences that offer a spatial resolution of 94 μm × 94 μm and a slice thickness of 200 μm created from 15 averages at an acquisition time of 33 min 45 s. Whereas these scanning parameters allowed for a reasonable SNR in order to evaluate changes in functional connectivity between key medullary nuclei in the baroreflex neural circuits, further improvements in resolution can only be met with difficulty. If we were to increase the spatial resolution to 47 μm × 47 μm and the slice thickness to 100 μm while maintaining the current SNR through averaging, the total acquisition time would be extended to approximately 36 h. This is unacceptable for a physiological study using live animals. Given that the average length of NTS neurons in mice is 10 μm, DTI analysis based on the scanning parameters currently used by us does not allow us to decide whether second‐order NTS neurons that are directly driven synaptically by the primary baroreceptor afferents are the projection neurons to the NA or CVLM or whether they are third (or higher) order NTS interneurons in the baroreflex circuits.

Partial rewiring of the neural circuit for baroreflex‐mediated sympathetic vasomotor tone

Absence of functional connectivity between NTS and CVLM

The contemporary dogma stipulates that the CVLM participates in baroreflex‐ mediated sympathetic vasomotor tone by acting as an inhibitory synaptic relay that connects the NTS to the RVLM (Ross et al. 1985; Ruggiero et al. 1989; Dampney, 1994; Spyer, 1994). One surprising finding from visualization of the baroreflex at work is the near absence of the obligatory functional connectivity between the NTS and CVLM. In an anterior‐posterior presentation of the FA map of the medulla oblongata in mice (Su et al. 2016) and rats (Tsai et al. 2017 b), we rarely encountered (6 out of 112 observations in mice; 15 out of 88 observations in rats; P < 0.0001, Chi‐square analysis) the presence of functional connectivity between the NTS and CVLM under basal conditions or in animal models of diseases. To comply with statutory regulations on experiments using laboratory animals, mice were under isoflurane anaesthesia during the MRI procedures. We therefore acknowledge that the absence of the NTS‐CVLM connectivity may be an artifact of anaesthesia, which suppresses sufficient action potential traffic to generate a DTI signal. This issue can only be resolved when MRI of animals in a conscious state becomes feasible. We recognize that the anaesthetic state of the animals will affect baroreflex and blood pressure control, as it affects central mechanisms of respiration, but we reason that this is not a concern because under the same experimental conditions, robust functional connectivity exists between the NTS and RVLM and exhibits prominent temporal alterations that parallel the commensurate changes in baroreflex‐mediated sympathetic vasomotor tone. These findings therefore necessitate entertaining the idea of partially rewiring the neural circuit depicted in the classical literature which includes CVLM as an intermediate between the NTS and RVLM (Dampney, 1994; Spyer, 1994).

We recognize that our observations are at variance with those reported by Macefield & Henderson (2010) using blood oxygen level‐dependent (BOLD) measurements in the brainstem of awake human subjects, in conjunction with recording of muscle sympathetic nerve activity (MSNA). These authors found that elevations in MSNA are associated with increases in BOLD signal intensity in the medullary site that corresponds to the human equivalent of the RVLM, and reciprocal decreases in signal intensity in the regions of the NTS and CVLM. Given the differences in imaging technique (DTI versus BOLD), spatial resolution (94 μm versus 1.5 mm), and magnet strength (9.4T versus 3T), a direct comparison of results from the two studies is not applicable. Suffice it to say, we wish to stress that functional connectivity measured using BOLD analysis only implies connection between remote neurophysiological events that may be associated (Friston et al. 1996). On the other hand, tractographic analysis offers visualization of connectivity, or the lack of it, between two nuclei with commensurate functional consequences.

Revised neural circuit for baroreflex‐mediated sympathetic vasomotor tone

Activation of the NTS by the primary afferents from the baroreceptors depresses the excitatory outputs from the RVLM to the sympathetic preganglionic neurons in the IML of the thoracic spinal cord (Spyer, 1994). As such, it is mandatory that, in the absence of the CVLM, the revised neural circuit for baroreflex‐mediated sympathetic vasomotor tone must sustain an inhibitory input from the NTS to the RVLM, which in turn lessens the tonic excitatory action of these premotor sympathetic neurons on vasomotor tone. There are two rewiring options to accomplish this stipulated goal. First, an excitatory projection from the NTS terminates on inhibitory interneurons that synapse on spinally projecting RVLM neurons (Fig. 1 C). Second, an inhibitory projection from the NTS terminates directly on RVLM neurons that project to the IML in the spinal cord (Fig. 1 D). Direct projection of barosensitive neurons in the NTS to the RVLM has been reported (Ross et al. 1985; Dampney & Horiuchi, 2003), as has the possibility of those NTS neurons synapsing with GABAergic interneurons in the RVLM (Meeley et al. 1985). On the other hand, direct GABAergic projection from the NTS to the RVLM is absent (Meeley et al. 1985). More importantly, given that an increase in FA between the NTS and RVLM accompanies a reduction in baroreflex‐mediated sympathetic vasomotor tone (Su et al. 2016), it appears that the rewired neural circuit depicted in Fig. 1 C is more akin to the functionality of this arm of baroreflex. Because of its provocative nature, this revised circuit must, of course, be subjected to further validation by sophisticated approaches such as optogenetics.

Lewis & Coote (1995) described a direct projection from the NTS to the IML of the spinal cord that they thought underpins the baroreflex sympatho‐inhibitory component. Verification of this NTS‐spinal pathway by tractography would have offered another alternative to the revised neural circuit for baroreflex‐mediated sympathetic vasomotor tone. Unfortunately, despite rigorous efforts, we have yet to successfully image this NTS‐IML connection with DTI. The existence of projections from the NTS to the RVLM has been documented since the 1980s (Ross et al. 1985; Hancock, 1988; Morilak et al. 1989; Van Bockstaele et al. 1989; Otake et al. 1992, 1993; Núñez‐Abades et al. 1993). Koshiya & Guyenet (1996) further proposed that this projection originates from a population of chemosensitive neurons in the caudal NTS that are devoid of respiratory modulation. We therefore acknowledge that the NTS‐RVLM connectivity identified by DTI could be mediating the chemoreflex and not necessarily the baroreflex. However, this issue can only be further addressed on development of DTI to a level of sophistication that allows for functional imaging that distinguishes baroreflex from chemoreflex pathways relaying through NTS.

Baroreflex functionality revealed by DTI – physiological implications

Cardiac vagal baroreflex

Tractographic analysis revealed the presence under physiological conditions of robust functional connectivity (Figs 4 and 5) between NTS and NA. In an in situ perfused working heart and brainstem preparation of the rat, which eliminates the consequences of anaesthetics on respiratory sinus arrhythmia (Bouairi et al. 2004), Farmer et al. (2016) reported that cardiac vagal motoneurons in the NA do not appear to possess any intrinsic autoactivity and rely on synaptic inputs (e.g. respiratory modulation) to adjust their discharges. Stimulation of the NTS evokes a glutamatergic pathway that activates both NMDA and non‐NMDA postsynaptic currents in cardiac vagal neurons (Wang et al. 2001). We are also cognizant that the vagus nerve exerts a tonic inhibitory action on the heart, although the origin of this classical phenomenon known as vagal brake has been elusive. We therefore interpret the NTS‐NA functional connectivity to suggest that the basal cardiac vagal baroreflex activity is sustained by a tonic excitatory input from the NTS to NA, which in turn exerts a tonic inhibitory action on the heart via the vagus nerve (Figs 4 and 5). Since the activity of the NTS is governed by afferent inputs from the baroreceptors, this interpretation provides a holistic view of the operation of the cardiac vagal baroreflex as an integrated feedback loop. It also offers a plausible explanation for the origin of the vagal brake. Most importantly, changes in this baseline functional connectivity between the NTS and NA dictate alterations in the functionality of this arm of the baroreflex.

Figure 4. Reversible disruption of functional connectivity in the baroreflex neural circuits.

Figure 4

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Tractographic analysis revealed the presence of robust functional connectivity (yellow arrows) between NTS and NA or RVLM under physiological conditions. The NTS‐NA functional connectivity suggests that the basal cardiac vagal baroreflex activity is sustained by a tonic excitatory input from the NTS to NA, which in turn exerts a tonic inhibitory action on the heart. Likewise, regardless of the synaptic arrangements as depicted in Fig. 1 C and D, the NTS‐RVLM functional connectivity suggests that basal baroreflex‐mediated sympathetic vasomotor tone is sustained by a tonic inhibitory input from the NTS to RVLM, which in turn lessens the tonic excitatory action of these premotor sympathetic neurons on vasomotor tone. Under pathophysiological conditions such as neurogenic hypertension, the efficacy of both arms of the baroreflex is by definition impeded. Thus, there is a decrease in the tonic excitatory influence of the NTS on NA that reduces the inhibitory actions of the vagus nerve on the heart or a decrease of the tonic inhibitory influence of the NTS on RVLM that increases the sympathetic vasomotor tone despite heightened neuronal impulses from the baroreceptor afferents. Tractographic analysis of the medulla oblongata revealed that this pathophysiological condition is created when the functional connectivity between the NTS and NA or RVLM is disrupted because of oxidative stress induced in the NTS and RVLM (shaded in yellow). Intriguingly, the disrupted functional connectivity is reversed on application of, for example, antioxidant to the NTS or RVLM, resulting in the resumption of NTS‐NA or NTS‐RVLM functional connectivity under physiological conditions. (Adapted from Su et al. 2016; no permission required.)

Figure 5. Irreversible disruption of functional connectivity in the baroreflex neural circuits.

Figure 5

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Under pathological condition such as hepatic encephalography, the initial response of the animals is physiological to sustain BP and HR. Over time, this physiological response gradually develops into a pathophysiological and thence to a pathological condition because of increasing nitrosative stress (formation of peroxynitrite) in the NTS and RVLM (shaded in red). However, the two arms of the baroreflex exhibit disparity both in modus operandi and time course. The baroreflex‐mediated sympathetic vasomotor tone proceeds from decline to disappearance, which corresponds to a change from disruption to ultimate severance of the NTS‐RVLM functional connectivity, signifying that brainstem death, a process that is irreversible, has ensued. On the other hand, with a reduced but maintained NTS‐NA functional connectivity, the functionality of cardiac vagal baroreflex is retarded but sustained, leading to the preservation of HR until the abrupt occurrence of cardiac death that follows brainstem death. (Adapted from Su et al. 2016; no permission required.)

Baroreflex‐mediated sympathetic vasomotor tone

Robust functional connectivity also exists between the NTS and RVLM. Guyenet et al. (2013) noted in their report that “the RVLM was found to contain spinally projecting neurons that were active at rest and inhibited by baroreceptor stimulation to a degree roughly commensurate with the change in sympathetic vasoconstrictor outflow”. As such, it is conceivable that, regardless of the synaptic arrangements depicted in Fig. 1 C and D, the basal baroreflex‐mediated sympathetic vasomotor tone is sustained by an overall tonic inhibitory input from the NTS to RVLM, which in turn lessens the tonic excitatory action of these premotor sympathetic neurons on vasomotor tone. Again, this interpretation provides a holistic view of the operation of the baroreflex‐mediated sympathetic vasomotor tone as an integrated feedback loop. It also reveals the existence of the equivalence of the vagal brake at the level of the RVLM in the regulation of sympathetic vasomotor tone. Likewise, and most importantly, changes in the baseline functional connectivity between the NTS and RVLM dictate alterations in the functionality of this arm of the baroreflex.

Reversible disruption of functional connectivity in the baroreflex neural circuits – pathophysiological implications

Under pathophysiological conditions when disruption of the functional connectivity between key nuclei in the baroreflex neural circuits is reversible, the associated disease condition is amenable to remedial measurements. One suitable illustrative example is neurogenic hypertension (Fig. 4), a chronic hypertensive state that is not induced by defects of peripheral organs or blood vessels, but is consequential to depressed baroreflex. A well‐established animal model for neurogenic hypertension entails intracerebroventricular infusion of angiotensin II (Ang II) by osmotic minipump in mice (Tsai et al. 2013) or rats (Chan et al. 2009). Under this experimental model, the efficacy of the cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone is by definition impeded. As such, despite heightened neuronal impulses from the baroreceptor afferents, there is a decrease in the tonic excitatory influence of the NTS on NA that reduces the inhibitory actions of the vagus nerve on the heart (withdrawal of the vagal brake) or a decrease in the tonic inhibitory influence of the NTS on RVLM that increases the sympathetic vasomotor tone (Fig. 4). Tractographic analysis of the medulla oblongata revealed that this pathophysiological condition of tachycardia and sustained hypertension is created when the functional connectivity between the NTS and NA and between the NTS and RVLM is disrupted because of oxidative stress induced by Ang II in the NTS and RVLM (Chan et al. 2005b , 2010; Tsai et al. 2013). Intriguingly, the disrupted functional connectivity is reversed on termination of Ang II infusion or when antioxidant or Ang II blocker is applied to the NTS (Tsai et al. 2013) or RVLM, resulting in the resumption of physiological conditions when HR, BP and cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone return to baseline. Baroreflex function is re‐set such that the baroreflex remains operative over a higher pressure range with a reduction in the reflex sensitivity. Baroreflex gain sensitivity seems to affect the cardiac vagal component more than the sympathetic vasomotor limb. Unfortunately, based on the current DTI scanning parameters, we have yet to image the changes in baroreflex gain sensitivity during neurogenic hypertension.

Irreversible disruption of functional connectivity in the baroreflex neural circuits – pathological implications

Fatality ensues when the pathophysiological condition advances to a pathological condition under which the functional connectivity between key substrates in the baroreflex circuits is irreversibly severed and the associated disease condition is no longer amenable to remedial measurements. One suitable illustrative example is hepatic encephalopathy (HE) (Fig. 5), a condition most commonly seen in patients suffering from advanced liver cirrhosis or severe hepatocellular dysfunction (Patel et al. 2012; Montano‐Loza, 2014), with a high mortality rate without liver transplantation (Munoz, 1993; Cash et al. 2010). A well‐established animal model for HE entails intraperitoneal administration of azoxymethane (AOM) in mice (Matkowskyj et al. 1999; Bélanger et al. 2006) or thioacetamide (TAA) in rats (Zimmermann et al. 1989; Butterworth et al. 2009). Under this experimental model, the initial physiological response that sustains BP and HR gradually advances to a pathophysiological and thence a pathological condition because of increasing nitrosative stress (formation of peroxynitrite) induced by AOM or TAA in the NTS and RVLM (Su et al. 2016; Tsai et al. 2017 b). However, the two arms of the baroreflex exhibit disparity both in mode of operation and time course (Fig. 5). The baroreflex‐mediated sympathetic vasomotor tone proceeds from decline to disappearance which corresponds to disruption to ultimate severance of the NTS‐RVLM functional connectivity, signifying that brainstem death (Kuo et al. 1997; Yien et al. 1997; Yen et al. 2000; Chan et al. 2005 a) has ensued during the final, pathological stage of HE, a process that is irreversible. On the other hand, with a reduced but maintained NTS‐NA connectivity, the functionality of cardiac vagal baroreflex is retarded but sustained, leading to the preservation of HR until the abrupt occurrence of cardiac death that follows brainstem death.

Plausible explanation for the time lapse between brainstem death and cardiac death

The interval when cardiac functions are sustained after brainstem death until the invariable asystole (cardiac death) is one of the driving forces for recognizing brainstem death as the legal definition of death in the US, UK and many other countries (Pallis, 1983; Pallis & Prior, 1983; Hung & Chen, 1995; Wijdicks, 1995; Haupt & Rudolf, 1999). This time window is vitally important for organ transplantation, which requires organ(s) retrieved from the donor to be in a functional condition. Our results from DTI provided direct evidence to support the notion that sustained functional connectivity between the NTS and NA in the face of severed NTS‐RVLM functional connectivity (Fig. 5), leading to the preservation of cardiac performance because of the unceasing cardiac vagal baroreflex, is responsible for the vital time lapse between brainstem death and the abrupt occurrence of cardiac death that inevitably follows.

Other forms of baroreflex functionality revealed by DTI

Anomalous baroreflex functionality is inherent in floxed and Cre‐Lox mice

The last two decades have seen the emergence of Cre‐Lox recombination as one of the most powerful and versatile technologies for cell‐specific genetic engineering of mammalian cells (Tsien, 2016). For investigators using this technique, the primary concern is whether the predicted genome (e.g. site‐ and cell‐conditional knockout) has been correctly modified, and the targeted phenotypes (e.g. loss of memory or behaviour) expressed. Rarely are the physiological conditions of the animals routinely examined because the general assumption is that they are normal. Based on corroborating results from radiotelemetric recording and DTI in BDNF‐floxed mice, we reported recently (Tsai et al. 2017 a) that despite comparable BP and HR with C57BL/6 or Cre mice in the conscious state, floxed and Cre‐Lox mice exhibit reduced NTS‐RVLM and NTS‐NA functional connectivity, coincidental with diminished baroreflex‐mediated sympathetic vasomotor tone and cardiac vagal baroreflex. The identification of anomalous baroreflex functionality inherent in floxed and Cre‐Lox mice points to the importance of incorporating physiological phenotypes into studies that engage in gene manipulations such as Cre‐Lox recombination.

Hypertensive side‐effect of anti‐vascular endothelial growth factor therapy (anti‐VEGF) therapy

As a potent angiogenesis stimulator, VEGF has been established as an efficacious clinical target for anticancer therapy (Meadows & Hurwitz, 2012). Unfortunately, the enthusiasm of using anti‐VEGF antisera or VEGF inhibitors is tempered by adverse cardiovascular effects, chief among which is hypertension (Mir et al. 2009). We found (Tsai et al. 2015) that adult male Flk‐1 (VEGFR2/KDR) deficient mice maintained as heterozygous (KDR+/−) colonies exhibit significant diurnal hypertension and tachycardia because of preferential disruption of the functional connectivity between NTS and NA that leads to withdrawal of the tonic baroreflex‐modulated vagal brake to the heart. We reasoned that since the reduced availability of Flk‐1 in the NTS of KDR+/− mice in effect mimics the pharmacological actions of anti‐VEGF agents, it is conceivable that anti‐VEGF therapies may engage the same pathophysiological mechanism to elicit the hypertensive side‐effects.

Concluding remarks

This review has summarised new evidence to support the notion that, in addition to morphological or pathological evaluations, tractographic analysis of the baroreflex neural circuits offers a hitherto unexplored research dimension to studies of the engagement of baroreflex in cardiovascular regulation. We showed that changes in FA between key nuclei in the baroreflex neural circuits are commensurate with the manifested functionality of this reflex. More intriguing is the observation that disrupted functional connectivity is reversible. This suggests that we are dealing with a physiological phenomenon that does not require physical damage such as demyelination or axonal injury. A corollary of this notion is the need to expand the definition of ‘functional connectivity’. Conventionally, this term is used specifically in conjunction with functional MRI, and is defined as the statistical association or dependency among two or more anatomically distinct time series while it is agnostic regarding causality or direction of connections (Friston et al. 1996). Our stipulation that an increase, decrease or disappearance of FA can be taken to infer augmentation, reduction or cessation of impulse traffic between two brain structures implies that this connectivity has a physiological component that fits within the connotation of ‘functional connectivity’. Under this newfound definition, disruption or severance of functional connectivity refers to reversible or irreversible cessation of impulse traffic without the necessary presence of physical damage to the axons.

The robust functional connectivity between the NTS and NA or NTS and RVLM revealed by DTI offers a holistic view of the modus operandi of the cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone. Since the activity of the NTS is primarily governed by afferent inputs from the baroreceptors, the activity of which depends on the prevailing BP, this functional connectivity can be taken to reflect the moment‐to‐moment feedback loops at work. Of special clinical relevance is the demonstration that changes in the baseline functional connectivity between the NTS and NA or RVLM dictate alterations in the functionality of the two arms of the baroreflex. In particular, under pathophysiological conditions when the disrupted functional connectivity is reversible, the associated disease condition is amenable to remedial measurements. On the other hand, fatality ensues on irreversible severance of the functional connectivity when the pathophysiological conditions evolve to pathological conditions. It follows that future design of new therapies may be guided towards either reversing a pathophysiological condition to a physiological condition, or preventing a pathophysiological condition from progressing to a pathological condition.

Despite its vital importance, the baroreflex is by no means a key research focus in contemporary medicine. The new clinical insights into baroreflex functionality gained from visualization of impulse traffic within the neural circuits of cardiac vagal baroreflex and baroreflex‐mediated sympathetic vasomotor tone under physiological, pathophysiological and pathological conditions may hopefully rekindle interest in this subject. Several new aspects of baroreflex functionality are additionally revealed by DTI. These include the absence of CVLM from the neural circuit for baroreflex‐mediated sympathetic vasomotor tone, the disparity in the time course of changes in NTS‐NA and NTS‐RVLM functional connectivity during brainstem death, the anomalous baroreflex functionality in Cre‐Lox mice, and the preferential disruption of the functional connectivity between NTS and NA in Flk‐1 deficient mice. We are cognizant that these developments, particularly the partially rewired neural circuit for baroreflex‐mediated sympathetic vasomotor tone, may be provocative in nature. Nevertheless, as science often moves fastest when ideas are challenged, on further validation, these new concepts possess the potential to alter our current views on brainstem cardiovascular regulation that bear clinical and therapeutic implications.

Additional information

Competing interests

None declared.

Author contributions

C.Y.T., J.Y.H.C. and S.H.H.C. conceived and designed the experiments cited in this review. C.Y.T., Y.Y.P. and S.H.H.C. carried out the experiments and data analyses. All authors contributed to interpretation of the data. C.Y.T. and Y.Y.P. drafted the manuscript. All authors contributed to critical revision and approved the final version of the manuscript.

Funding

DTI studies by the authors cited in this review were supported in part by the Ministry of Science and Technology, Taiwan (MOST‐106‐2320‐B‐182A‐014‐MY3 for C.Y.T.; MOST‐98‐2923‐B‐182A‐001‐MY3, MOST‐100‐2321‐B‐182A‐008, MOST‐103‐2321‐B‐182A‐001 and MOST‐103‐2321‐B‐182A‐016‐MY3 for S.H.H.C.), and the Chang Gung Medical Foundation, Taiwan (CMRPG890312 for Y.Y.P.; OMRPG8C0051 for J.Y.H.C.; and OMRPG8C0021 for S.H.H.C.).

Acknowledgements

We thank Ms Jacqueline C.C. Wu for producing the elegant artwork for Figs 2 and 3.

Biographies

Ching‐Yi Tsai is an Assistant Professor in the Institute for Translational Research in Biomedicine. Her research interests are primarily on signalling cascades and neural circuits involved in brainstem cardiovascular regulation associated with brainstem death and neurogenic hypertension.

graphic file with name TJP-597-41-g001.gif

Yan‐Yuen Poon is an Assistant Professor in the Department of Anesthesiology. His research interest is on spinal mechanisms of pain and antinociception.

Julie Y. H. Chan is Chair Professor in the Institute for Translational Research in Biomedicine and current President of the International Union of Physiological Sciences. Her research focuses on molecular and cellular mechanisms underlying oxidative stress‐mediated cardiovascular disorders, particularly with reference to neurogenic hypertension and metabolic syndromes.

Samuel H. H. Chan is Distinguished Chair Professor and Director of the Institute for Translational Research in Biomedicine. His research focuses on central cardiovascular regulatory functions, particularly in translational research on brainstem death and neurogenic hypertension.

Edited by: Ole Petersen & Benedito Machado

All authors are affiliated with the Chang Gung Memorial Hospital in Taiwan.

Contributor Information

Julie Y. H. Chan, Email: jchan@adm.cgmh.org.tw

Samuel H. H. Chan, Email: shhchan@adm.cgmh.org.tw.

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