CNSNT-Gu Xiaoping's team revealed that enhancing astrocyte networks can improve brain network abnormalities and cognitive dysfunction caused by long-term isoflurane anesthesia

Written by - Yuqiang Han, Rui Dong Editor-in-Charge - Sizhen Wang—Xia Ye's
perioperative neurocognitive disorder (PND) is a major complication characterized by impaired memory, learning, and executive function in the short and long term after surgery and anesthesia, causing disability and pain in millions of patients each year[1].

At present, the specific molecular mechanism of the onset of PND is unknown
.
Theoretically, anesthetics should cause temporary, reversible loss of consciousness and reactivity without any additional side effects
.
However, numerous studies have found that long-term exposure to isoflurane may lead to cognitive dysfunction [2].

Previous studies of PND have focused on microscale or syncytium scale,[3] but very little
has been done at mesoscale.
So-called mesoscale studies refer to the association
between changes in functional connections between different brain regions of the brain and PND.
With the development of neuroimaging technology, conditions have been created for the study of brain function at the mesoscale [4-6].

Currently, mainstream neuroimaging techniques include resting functional magnetic resonance (rs-fMRI) and manganese ion-enhanced functional magnetic resonance (MEMRI
).

Astrocytes in the central nervous system are interconnected cellular networks made up of gap junctions (GJs), which consist primarily of channel protein nemonin 43 (Cx43) [7].

Astrocytes sense neuronal activity in the form of a network that regulates neural activity and maintains homeostasis in the brain [8].

In some pathological cases, a GJS breaks to form a half-channel of two Cx43 molecules, called GJs-Cx43 decoupling
.
Previous studies have shown that long-term isoflurane exposure can lead to GJS-Cx43 uncoupling, leading to the occurrence of PND [8].

However, the effect of GJS-Cx43 uncoupling on functional connections between different brain regions of the brain remains unclear
.

Recently, the team of Professor Gu Xiaoping of the Drum Tower Hospital affiliated to Nanjing University School of Medicine and the team of Professor Zhang Bing published a report entitled "Enhancement of astrocytic gap junctions connexin43 coupling can improve long-term" at CNS Neuroscience and Therapeutics (CNSNT).
In the article "isoflurane anesthesia mediated brain network abnormalities and cognitive impariment", Dong Rui, Lü Pin and Han Yuqiang were co-first authors, and Professor Gu Xiaoping, Professor Zhang Bing, and Associate Professor Xia Jiao were co-corresponding authors
。 PND has been found to be a brain network disorder that includes brain regions associated with sleep-wake rhythms, brain regions associated with background memory and fear memories, the hippocampus-amygdala circuit, the septum-hippocampus circuit, and the internal olfactory-hippocampal circuit
.
By increasing the expression of GJs-Cx43, the effect of reconfigurable PND on the network of astrocytes partially reversed the abnormalities
in the above circuits.
In addition, the main features of PND pathology are oxidative stress and neuroinflammation to damage to the hippocampus
.
The study in this paper suggests that promoting the formation of GJS-Cx43 can also improve cognitive dysfunction caused by long-term isoflurane anesthesia
by alleviating oxidative stress in the hippocampus.
(Further reading: Gu Xiaoping's team's latest research results, see the "Logical Neuroscience" report (click to read): J Neuroinflammation - Gu Xiaoping's research group reveals the important role of astrocyte networks in long-term isoflurane anesthesia-mediated postoperative cognitive dysfunction)

First, the researchers divided C57BL/6 mice into three groups, control group (Con+V), Isoflurane anesthesia group (Iso+V) and ZP1069 dosing group (Iso+D
).
Con+V did not give any intervention, Iso+V underwent a 6 h isoflurane anesthesia intervention to establish a behavioral model of PND, and Iso+D underwent drug therapy with the gap nebulin enhancer ZP1069 drug after administering 6H isoflurane anesthesia (Figure 1).

。 The behavioral results found that compared with Con+V, long-term isoflurane anesthesia led to a significant decrease in the learning and memory ability of mice on days 1 and 3 after anesthesia, and after the treatment of ZP1069, the learning memory of mice was improved (Figure 2), which suggested that the use of ZP1069 to enhance GJs-Cx43 coupling can alleviate cognitive dysfunction caused by long-term isoflurane anesthesia
.
Figure 1 Time plot
of groups, treatments, and procedures used in magnetic resonance imaging (MRI) studies.
(Source: Rui Dong et al.
, CNSNT, 2022) Figure 2 Enhanced foliage conjugation of crevice 43 can alleviate cognitive dysfunction caused by long-term isoflurane anesthesia (Source: Rui Dong et al.
, CNSNT, 2022)
In order to further explore the activity changes of different brain regions in PND mice, the authors used MEMRI to explore the changes in the activity of each brain region under the PND model (Figure 1A).

。 To rule out confounding factors, the authors first analyzed the brain, thalamus, and hippocampus total volume of the mice, and found no statistical difference (Figure 3A
).
The results of voxel-based MEMRI whole brain analysis showed that the change of whole brain signal intensity caused by long-term isoflurane anesthesia was specific to the brain region, and the trend of MEMRI signal intensity change in different brain regions was not completely consistent (Figure 4A-C).

Overall, the trend of MEMRI signal strength change in Iso+V vs.
Con+V is the opposite of
the trend in Iso+D vs.
Iso+V.

Figure 3 Comparing mouse brain volume and MnCl2 absorption rate (Source: Rui Dong et al.
, CNSNT, 2022) In order to better understand the relationship between these brain regions and PND, the researchers extracted regions of interest (ROI)
based on voxel results for statistical analysis
.
The analysis results suggest that there is a statistical difference between 28 brain regions between 3 groups of mice (Figure 4D
).
Subsequently, the researchers divided the 28 brains into Class 4, Class I: Reverse Activation, i.
e.
, enhanced GJs-Cx43 coupling reversible the decrease in MEMRI signal intensity caused by long-term isoflurane anesthesia; Category II: reverse inhibition, which means that long-term isoflurane anesthesia causes increased MEMRI signal strength, but enhanced GJs-Cx43 coupling reverses; Class III: Activation enhancement, that is, there is no statistically significant difference in MEMRI signal strength between the Con+V group and the Iso+V group, while the MEMRI signal strength in the Iso+D group increases; Category IV: No effect, i.
e.
, enhanced GJs-Cx43 coupling has no effect on changes in MEMRI signal intensity caused by long-term isoflurane anesthesia (Figure 4D).

These results suggest that the cognitive dysfunction caused by isoflurane is a complex network rather than a region-based disease, and that GJS-Cx43 uncoupling also involves multiple brain regions
.

Fig.
4 Voxemotive whole brain and region-based classification analysis of manganese-enhanced magnetic resonance imaging (MEMRI) under different interventions (Source: Rui Dong et al.
, CNSNT, 2022)
In order to further understand the functional connections between differential brain regions, the researchers selected the differential brain regions in MEMRI as seed regions (ROI), and used fMRI for functional connection analysis of these differential brain regions (Figure 5).

。 The results of the study showed that compared with the Con+V group, there were 154 connections above the middle line and 224 connections below the center line in the Iso+V group, which showed that the connection strength of the Iso+V group was significantly reduced
.
Compared with Iso+V, there are 240 connections above the midline and 138 connections below the midline in the Iso+D group, which means that the Iso+D group improves the functional connections
between brain regions compared to the Iso+V group.
At the same time, it is worth noting that compared to the Con+V group, the Iso+D group also showed an increase in functional connections (225 connections above the center line and 153 connections below the center line).

This suggests that long-term isoflurane exposure can lead to a decrease in FC strength, while promoting GJS-Cx43 coupling can partially reverse dysfunctional connectivity abnormalities caused by long-term isoflurane anesthesia, accompanied by compensatory changes
in functional connections in other brain regions.

Fig.
5 Functional connection analysis of brain regions with manganese enhanced magnetic resonance imaging (MEMRI) differential signal (Source: Rui Dong et al.
, CNSNT, 2022)
The hippocampus region is considered to be a key brain region in the pathophysiology of PND, mainly involved in the formation
of learning and memory.
The authors found long-term isoflurane anesthesiaAfter that, the MEMRI signal strength in the hippocampus is significantly reduced
.
When ROI-based analysis was performed with the hippocampus as the ROI, the results showed that functional connections in some areas of the Iso+V group were significantly reduced compared to the Con+V group, and the study also found that the reduction of these functional connections could be reversed by ZP1069 (Figure 6).

Further analysis found that compared with Iso+V, ZP1069 not only has a reversal effect, but also significantly increases the functional connections
between some hippocampal brain regions.
The above results suggest that long-term isoflurane anesthesia can lead to abnormal functional connections between the hippocampus region and other brain regions, and these abnormal changes can be reversed
by reconstructing the GJs-Cx43-mediated network of astrocytes.
There is no doubt that these differential functional connections have important implications
for the next stage of neural circuit research.

Figure 6 Whole brain functional connectivity analysis with the hippocampus region as the seed region (Source: Rui Dong et al.
, CNSNT, 2022)
To explore the potential mechanism by which GJs-Cx43 uncoupling may affect the brain network of PND mice, the authors examined the levels of oxidative stress and neuroinflammation in the hippocampus (Figure 7A-E).

The results showed that ZP1069 significantly increased the GJs-Cx43 coupling in the hippocampus, but did not increase the overall Cx43 level
.
Further studies found that increasing GJs-Cx43 coupling in the hippocampus region can significantly reverse hippocampal nerve inflammation, increase superoxide dismutase activity and reduce malondialdehyde and ROS levels
.

Figure 7 Increasing the coupling of gap junction protein 43 (GJS-Cx43) alleviates oxidative stress and neuroinflammation in the hippocampus region and primary cultured astrocytes (Source: Rui Dong et al.
, CNSNT, 2022)
In order to further explore the effect of isoflurane on GJs-Cx43-mediated astrocyte coupling, the authors took purified primary astrocytes as the research object
。 After the 6H anesthesia intervention was given to primary astrocytes, it was found that the total Cx43 of primary astrocytes treated with isoflurane was significantly reduced, and there was a significant decoupling of GJs-Cx43, an increase in IL-1β and IL-6, a significant inhibition of superoxide dismutase activity, and a significant increase
in the content of malondialdehyde and ROS 。 After the ZP1069 intervention, ZP1069 increased GJs-Cx43, reduced IL-1β and IL-6 expression, reversed the reduction of superoxide dismutase activity and reduced the content of malondialdehyde and ROS without affecting the total Cx43 expression (Figure 7 F-J).

。 These results suggest that long-term isoflurane exposure causes GJs-Cx43 to decouple in primary astrocytes and induces oxidative stress and neuroinflammation, while rebuilding the astrocyte network by increasing GJS-Cx43 coupling can alleviate isoflurane-induced astrocyte oxidative stress and inflammation
.

First, the study combined with magnetic resonance technology, molecular biology, cytology and animal behavior to clarify the relationship between
the astrocyte network mediated by slit junction protein 43 (Cx43) and abnormal brain activity mediated by perioperative neurocognitive disorder (PND).
Previous studies have found that the uncoupling of astrocyte networks leads to reduced CA1 neuronal activity and long-term synaptic plasticity [9], which the authors suggest may be related to
oxidative stress and neuroinflammation in astrocyte uncoupling.
Notably, previous clinical studies have shown that reductions in thalamus and hippocampus volume are strongly associated with PND [10], however, the results of this study suggest that there is no change
in the volume of the whole brain, thalamus, and hippocampus in mice with PND.

In this article, the authors report on some of the brain regions affected by PND, and this part of the differential brain region seems to be used to explain the effects of isoflurane on
animal behavior.
For example, it has been previously reported that rhesus monkeys observed a decrease in intimate social behavior and an increase in anxiety-related behavior after 5 hours of receiving isoflurane anesthesia [11].

The authors also found in the magnetic resonance results of the PND model that MEA[12], the brain region responsible for emotional or social behavior, had a significant decrease in MEMRI signal intensity after a long period of anesthesia with isoflurane and could not recover
by increasing gap junctions (GJs)-Cx43 coupling.
This suggests that isoflurane may influence social behavior by inhibiting the activity of MEA, however, the neural circuit mechanisms employed by it need to be elucidated
in subsequent studies.

In summary, the authors' research data suggest that increasing GJs-Cx43 coupling can reduce neuroinflammation and oxidative stress, improve abnormal brain function network patterns caused by long-term isoflurane anesthesia, and help restore potential neural circuits associated with PND.
This comprehensive experimental approach also provides new insights into
the changes in brain function associated with astrocytes at the mesoscale level.

Original link: https://pubmed.
ncbi.
nlm.
nih.
gov/36153812/
corresponding authors: Gu Xiaoping (first from left), Zhang Bing (second from left), Xia Jiao (third from left); Co-first authors: Dong Rui (third from right), Lü Pin (second from right), Han Yuqiang (first from right) (photo provided by: Gu Xiaoping's research team at Affiliated Drum Tower Hospital of Nanjing University School of Medicine)
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End of article