The pancreas is densely innervated, and neural signals play a significant role in glucose regulation by modulating pancreatic hormone release. However, relatively little is known about the anatomical relationships between islets and nerves across the whole pancreas. In this webinar, Dr. Sarah Stanley and Dr. Alexandra Alvarsson will discuss their research using tissue clearing and whole organ imaging of the pancreas to identify the 3D structure of pancreatic nerves and islets. In particular, they will provide an overview of their methodology, which provides detailed information and quantification of pancreatic innervation in healthy pancreas, in canonical models of diabetes and in samples from nondiabetic and diabetic donors. They will also present their findings, demonstrating greatly enriched innervation in the islets with regional variations. They will also discuss beta cell innervation in mouse models of diabetes and in pancreata from human donors with type 2 diabetes. Key Topics Include: - Tissue clearing and 3D imaging to allow the mapping of nerves in peripheral organs - Innervation of peripheral organs such as the pancreas - How pancreatic nerves are remodeled in diabetes

1. Remodeling
of pancreatic
innervation
in diabetes
Dr. Sarah Stanley
Dr. Alexandra Alvarsson
Diabetes, Obesity and
Metabolism Institute

2. Remodeling
of pancreatic
innervation
in diabetes
Dr. Sarah Stanley and Dr. Alexandra
Alvarsson discuss the use of whole-organ
imaging of the pancreas to reveal close
interactions between nerves and islets
and dynamic regulation of islet
innervation in diabetes.

3. Dr. Sarah Stanley
Dr. Alexandra Alvarsson
Diabetes, Obesity and
Metabolism Institute
Remodeling of
pancreatic innervation
in diabetes

4. • Consultant to Redpin Therapeutics
• Patent and patent applications
US patent WO 2016/049031 “Compositions and Methods to
Modulate Cell Activity”
US patent application PCT/US15/51457 “Compositions and Methods
to Modulate Cell Activity”
Disclosure

5. Excess and privation

6. • Glucose is virtually the sole fuel for the brain
• Brain consumes 60-70% of whole body glucose use in the resting state
Glucose-6-phospate
Hexokinase
1
Glucos
e
GLU
T1
GLU
T3
Glucose
Hexokinase
1
Endothelial
cells
Pericytes
Astrocytes
Neuron
The CNS relies on glucose as a fuel

7. Beta cell
Alpha cell
insulin
Glucago
n
Islet Glucos
e
excite
d
?
Glucos
e
inhibit
ed
?
Pancreas Brain
Specialized neurons act as glucose sensors

8. Lateral Arcuate
13% GE
1% GI
Nucleus of
solitary tract
10% GE
8% GI
Medial Arcuate
4% GE
14% GI
Paraventricular
nucleus
19% GE
8% GI
Ventromedial
hypothalamus
13-14% GE
8-16% GI
Dorsomedal
hypothalamus
15% GE
14% GI
Lateral
hypothalamus
20% GE
27-38% GI
Glucose-sensing neurons form a
distributed network

9. CNS regions sensing glucose connect to
autonomic outflow
0
2
4
6 0
2
4
6
0
2
4
6
8 - 2 - 4
4 2 0 -2 - 4 - 6 - 8
Bregma
Bregma
0
2
4
6 0
2
4
6
0
2
4
6
8 - 2 - 4
4 2 0 -2 - 4 - 6 - 8
Bregma
Bregma
PVT
PVH
ARC
DMH
VMH
Amygdala
LH
DMV
NTS
AP
D
C
B
E
A B C D
E
IML
RVLM
PARASYMPATHETIC
EFFERENTS
SYMPATHETIC EFFERENTS
Rostral
ventrolate
ral
medulla
Intermedi
o
lateral
nucleus
Dorsal
motor
nucleus of
vagus

10. • Increase glucagon
release
• Suppress insulin
release
• Altered insulin
sensitivity
• Reduced β cell
proliferation and
mass
Autonomic nerves innervate organs critical to
glucose regulation
• Increase insulin
release
• Increase β cell
proliferation and
mass
Parasympathetic Nervous System Sympathetic Nervous System

11. 11
Human studies
• Regional CNS activation with
hypoglycemia
• Cephalic phase insulin
release
• Altered glucose metabolism
with deep brain stimulation
and TCM
• Abnormal glucose tolerance
after vagotomy
• Abnormal response to
exercise and hypoglycemia
post pancreatic transplant
Neural regulation of glucose in humans
Rare pathologies
• Hypothalamic pathology
o high insulin levels
o insulin resistance
o impaired glucose
tolerance.
• Glucose abnormalities in
patients with demyelination
• Autonomic neuropathy
impairs hypoglycemic
responses

12. Spinal Sensory
Sympathetic efferent
Parasympathetic sensory
Nodose ganglion
Dorsal root
ganglia
Coeliac
ganglia
Intrapancreatic
ganglia
Multiple Inputs into pancreatic innervation
Parasympathetic efferent
Enteroendocrine
Dorsal motor nucleus
of vagus

13. Parasympathetic Inputs into the pancreas
Parasympathetic efferent
Dorsal motor nucleus
of vagus
• Cell bodies in dorsal motor
nucleus of vagus
• Anterograde tracing shows
inputs to intrapancreatic ganglia
(rat)
Berthoud and Powley 1991
• Vagal activation induces c-fos in
10-30% of intrapancreatic
ganglia (rats)
Wang et al 1999

14. Sympathetic Inputs into the pancreas
• Preganglionic neurons in
intermediolateral column project
to coeliac ganglia in splanchnic
nerves
Quinson et al 2001
• Sympathetic post-ganglionic
fibers project to intrapancreatic
ganglia, islets, vasculature and
lymph nodes.
Sympathetic efferent
Coeliac
ganglia

15. Sensory innervation of the pancreas
• Extensive sensory innervation
Fasanella et al 2008
• Vagal sensory innervation
involves exocrine and endocrine
pancreas
• Primarily chemosensors
Makhmutova et al, 2020
• Spinal sensory innervation from
T5 – T13
• Express substance P, TRPV1
and CGRP
Fasanella et al 2008
• Both mechano- and
chemosensors
Schloithe et al, 2008
Spinal Sensory
Parasympathetic sensory
Nodose ganglion
Dorsal root
ganglia

16. Intrapancreatic ganglia
• Mesh of intrapancreatic neurons
and intrapancreatic ganglia
• Often adjacent to islets to form
neuro-insular complexes
Tang et al, 2018
• Primarily cholinergic cell bodies
and fibers with neuropeptide
expression
De Giorgio et al, 1992

17. Imaging pancreatic innervation in 2 dimensions
• Extensive investigation of
pancreatic islet structure and
innervation using
immunohistochemistry
Chien et al, 2016
Parween et al, 2016
Fowler et al 2018
• Defined extensive
parasympathetic and
sympathetic innervation in
rodent islets.
• Sparse, primarily sympathetic
innervation in human islets
Rodriguez-Diaz et al, 2012
Spinal Sensory
Sympathetic efferent
Parasympathetic sensory
Nodose ganglion
Dorsal root
ganglia
Coeliac
ganglia
Intrapancreatic
ganglia
Parasympathetic efferent
Enteroendocrine
Dorsal motor nucleus of
vagus

18. Imaging pancreatic innervation in 2 dimensions
PRO
• Allow high resolution imaging
particularly of fine innervation
• Identify target structures
CON
• Highly heterogenous organ
• Laborious serial sectioning
• Tracing over long distances
difficult
• May miss regional differences
Insulin/TH
Insulin/synapsin
Dapi/synapsin
Immunohistochemistry in 2D
3D imaging

19. Imaging pancreatic innervation in 3 dimensions
• Recent resurgence in optical
clearing methods examining
pancreatic structure.
Richardson et al, 2015
• Advances in volumetric imaging
e.g. lightsheet microscopy,
optical projection tomography,
swept confocal aligned planar
excitation (SCAPE) microscopy.
• Progress 3D volumetric image
processing software advances
e.g. Image J, Matlab,
Neurolucida, Imaris etc.
2
0
Fructose
Ethyl cinnamate
Insulin/TH/vasculature

20. Imaging pancreatic innervation in 3 dimensions
Examination of sympathetic innervation in diabetic mice
• Fluorescent lectins and immunolabeling to examine vasculature and innervation
• Focusclear tissue clearing and confocal imaging
• Increased intra-islet sympathetic fibers associated with vasculature in STZ but not
NOD mice Chiu et al, 2012
Insulin/Vasculature/TH

21. Imaging pancreatic innervation in 3 dimensions
Examination of innervation and intrapancreatic ganglia in db/db mice
• Immunolabeling to examine innervation and islets
• RapiClear tissue clearing and tiled confocal imaging
• Identified intrapancreatic ganglia, increased connections with increasing size
• Increased pancreatic sympathetic innervation in db/db mice Tang et al, 2018a

22. Imaging pancreatic innervation in 3 dimensions
Examination of innervation in human pancreatic tissue
• Sympathetic innervation of islet vasculature and islet core
• Parasympathetic innervation of islet core Tang et al, 2018b
• Sensory innervation (SubP+) of intrapancreatic ganglia, not islets Chien et al,
2019
• Sympathetic innervation of islets, not exocrine pancreas, reduced in autoAb+
individuals compared to T1D. Campbell-Thompson et al, 2021

23. AIMS
To use optical clearing by iDISCO+ combined with
advanced 3D rendering to determine the distribution
of islets and innervation throughout the whole
pancreas in healthy animals, in mouse models of
diabetes, and in human donors without and with
diabetes.
23

24. • Duodenal vs. splenic region
C57BL/6N mice
• Nondiabetic vs. diabetic state
Non-Obese Diabetic (NOD) mice (NOD/ShiLtJ females, 12 - 16 weeks)
- Spontaneously develop type 1 diabetes with insulitis
- Two consecutive blood glucose measurements of >300 mg/dl  Diabetic
- Littermates with blood glucose <200 mg/dl  Nondiabetic controls
- Average nonfasting blood glucose:
115 ± 4 mg/dl (Nondiabetic) and 495 ± 62 mg/dl (Diabetic)
Multiple low-dose Streptozotocin (STZ)-treated mice (C57BL6/6N males,10 weeks)
- Progressive model of type 1 diabetes (beta cell toxin, insulitis)
- Treated with STZ (40 mg/kg) in citrate-saline buffer (pH 4.5) for five consecutive days
- Sacrificed at 5 or 15 days following the final STZ injection.
- Non–STZ-treated littermates were used as controls
- Average nonfasting blood glucose:
123 ± 9 mg/dl (Controls), 259 ± 18 mg/dl (Day 5), and 430 ± 17 mg/dl (Day 15)
Human donors without and with type 2 diabetes
AIMS

25. 1. Tissue harvesting
Nondiabetic
and diabetic
mice
• Heparinized saline
• 4% PFA (ice cold)
Alvarsson et al., Bio-Protocol,
2021

26. 2. Optical clearing by iDISCO+ Alvarsson et al., Bio-Protocol,
2021
Adapted from Renier et al., 2016
Pretreatment
Duration: 2 days
 Dehydration (6h)
 Delipidation (1h)
 Washing (methanol, 1.5h)
 Bleaching (o/n)
 Rehydration (2h)
 Permeabilization (1h+o/n)
 Washing (PTxwH,1.5h)
Immunolabeling
Duration: 10-16 days
• Blocking (o/n)
• Primary (3-6 days)
• Washing (PTxwH, 1 day+o/n)
• Secondary (3-6 days)
• Washing (PTxwH, 1 day+o/n)
• Washing (PBS, 1 day)
• Agarose embedding
Clearing
Duration: 1 day
• Dehydration (5h)
• Washing (methanol, 1.5h)
• Washing (DCM, 1.5h)
• Clearing in DBE
Remove soluble and
hydrophobic lipids
Reduce tissue
autofluorescence
• Insulin (beta cells)
• Glucagon (alpha cells)
• NF200 (pan-neuronal)
• TH (sympathetic)
• VAChT (parasympathetic)
Modified PtxwH buffer to improve
neuronal labelling (Alvarsson et al., 2020)
Samples immersed,
stored and imaged in
DBE
Refractive index: 1.562

27. 3. Imaging & Analysis Alvarsson et al., Bio-Protocol,
2021
Lightsheet
microscopy (1.3x, 4x,
12x)
Confocal microscopy
(10x)
Before
After
• 3D rendering
• Quantifications
• Volumetric
analyses
• Distance analyses

28. Imaging considerations Alvarsson et al., Bio-Protocol,
2021
Islet distribution
across
hemipancreata
Innervation
Ganglia,
alpha and
beta cells

29. 29

30. ANIMAL MODELS
30
C57Bl/6
mice
NOD
mice
STZ
mice
1. Healthy adult control mice (males)
2. Non-obese diabetic (NOD) mice,
spontaneously develop T1D and
insulitis (females)
3. Streptozotocin (STZ)-induced
mice, 5 low dose STZ injections
(males)

31. 31
T: Total
D: Duodenal
S: Splenic
REGIONAL VARIATION IN ISLET
CHARACTERISTICS IN CONTROL MICE
Islet
volume
Islet
density
Insulin
conten
t
Regional variation in islet volume
and insulin content
C57Bl/6
mice
NOD
mice
STZ
mice

32. 32
C57Bl/6
mice
NOD
mice
STZ
mice
Dramatic loss of islet volume and
density in diabetic NOD mice
(greater effect in S).
Islet
volume
Islet
density
Insulin
conten
t
REGIONAL VARIATION IN ISLET
CHARACTERISTICS IN NOD MICE

33. 33
C57Bl/6
mice
NOD
mice
STZ
mice
Low-dose STZ treatment reduces islet
volume and insulin content, but not the
islet density
Islet
volume
Islet
density
Insulin
conten
t
REGIONAL VARIATION IN ISLET
CHARACTERISTICS IN STZ MICE

34. Beta cell volume and islet
numbers highly variable.
The average islet volume lower in
diabetic donors.
Islet number per mm3 greater than
mice, but beta cell volume (%) was
similar.
Islet
volume
Islet
density
REGIONAL VARIATION IN ISLET
CHARACTERISTICS IN HUMANS

35. Control mice NOD mice STZ mice Human donors
ISLET VOLUME DISTRIBUTION
A shift towards smaller islets in human type 2 diabetes

37. GLUCAGON IN NOD MICE
Increased glucagon to insulin
ratio in diabetic NOD mice

38. GLUCAGON IN STZ MICE
Low-dose STZ
treatment reduced
glucagon volume and
increased glucagon to
insulin ratio

40. Regional variation in
endocrine innervation
in control mice
40
Islet innervation shows
regional variation in C57BL/6
mice.
Islet innervation
µm3/islet % of islet volume

41. 41
Islet innervation
µm3/islet % of islet volume
Exocrine
innervation
Regional variation in
endocrine innervation in
control mice

42. 42
Islet innervation (%) increased
in diabetic NOD mice,
particularly in the splenic
pancreas.
Islet innervation
µm3/islet % of insulin volume
Regional variation in
endocrine innervation in
NOD mice

43. 43
Islet innervation
µm3/islet % of glucagon volume
% of insulin volume
Regional variation in
endocrine innervation in
NOD mice

44. 44
Low-dose STZ treatment increases
endocrine innervation, particularly in
the splenic pancreas.
µm3/islet
Islet innervation
% of insulin volume
Regional variation in
endocrine innervation in
STZ mice

45. 45
Islet innervation
µm3/islet % of insulin volume % of glucagon volume
Regional variation in
endocrine innervation in
STZ mice

46. 46
NF200+ endocrine innervation
highly variable
The endocrine nerve volume (%)
was greater in samples from
diabetic individuals
Regional variation in
endocrine innervation in
humans
Islet innervation
µm3/islet % of islet volume

47. Regional variation in
endocrine innervation in
humans
No enrichment in NF200+
endocrine innervation compared to
exocrine tissue.
Islet innervation
µm3/islet % of islet volume

48. Control mice
ENDOCRINE NERVE DISTANCE
Insulin
Ins+ islets located <1.6
um from innervation:
Total: 6%
Duodenal: 6.5%
Splenic: 6%

49. NOD mice
ENDOCRINE NERVE DISTANCE
Insulin Glucagon
Alpha cell clusters
located <1.6 um from
innervation:
Nondiabetic: 15%
Diabetic: 34%
Ins+ islets located <1.6
um from innervation:
Nondiabetic: 15%
Diabetic: 10%

50. STZ mice
ENDOCRINE NERVE DISTANCE
Insulin Glucagon
Alpha cell clusters
located <1.6 um from
innervation:
Control: 26%
Day 5: 17%
Day 15: 22%
Ins+ islets located <1.6
um from innervation:
Control: 6%
Day 5: 6%
Day 15: 8%

51. Human donors
ENDOCRINE NERVE DISTANCE
Insulin
Ins+ islets located <1.6
um from innervation:
Control: 11%
Type 2 Diabetes: 29%
Altered nerve /
endocrine cell
associations in
diabetes?

53. GANGLIA AND ENDOCRINE NERVE CONTACTS
3D analysis of intrapancreatic NF200+ ganglia in IMARIS
Alvarsson et al., Bio-Protocol, 2021
• Number of ganglia per mm3
• Average ganglion volume
• Average distance between ganglia and insulin+ islets

54. GANGLIA AND ENDOCRINE NERVE CONTACTS
Alvarsson et al., Bio-Protocol, 2021
Distance analysis of α and β cell nerve contacts in IMARIS
• The Imaris Distance Transform Matlab XTension was used to
determine distance between NF200+ nerves and individual α or β
cells
• A distance of 0 was used to indicate a nerve contact

55. Intrapancreatic ganglia in
control mice
No regional variation in number,
volume or islet distance of NF200+
ganglia in control mice

56. Endocrine nerve
contacts in control mice
No regional variation in beta cell
nerve contacts

57. Intrapancreatic ganglia in
NOD mice
Preserved number and volume, but
significantly prolonged distance
between NF200+ intrapancreatic
ganglia and islets due to islet loss.

58. Endocrine nerve
contacts in NOD mice
No difference in alpha and beta
cell nerve contacts between
nondiabetic and diabetic NOD
mice
More alpha cell vs. beta cell
contacts

59. Intrapancreatic ganglia in
STZ mice
Preserved number and islet
distance, but reduced volume of
NF200+ ganglia with STZ-induced
diabetes.

60. Endocrine nerve
contacts in STZ mice
No difference in alpha and beta
cell nerve contacts between
nondiabetic and diabetic STZ
mice
More alpha cell vs. beta cell
contacts

61. Intrapancreatic ganglia in
human donors
Preserved number,
volume and islet
distance of NF200+
ganglia

62. Endocrine nerve
contacts in human
donors
No difference in beta cell nerve
contacts between nondiabetic
and diabetic human donors

64. TH in control mice
No significant regional
variation in TH+
exocrine and endocrine
innervation

65. VAChT in control mice
No significant regional
variation in VAChT+
exocrine and endocrine
innervation

66. 66
CONCLUSIONS
1. Innervation is highly enriched (10-fold) in the mouse endocrine
pancreas, with significant differences between duodenal and
splenic regions.
2. There is a close association between nerves and islets in both
mouse and human pancreata, which is maintained in diabetes.
3. Islet innervation was enriched (2-fold) in the pancreas of diabetic
NOD mice, and also during the progression of T1D induced by
STZ treatment (2-fold).
4. Islet innervation was also enriched in pancreata of human donors
with type 2 diabetes.

67. 67
• Cause of neuronal remodeling in diabetes
• Consequences of neuronal remodeling in diabetes
• Contribution of sympathetic, parasympathetic and sensory
innervation in diabetes
FUTURE DIRECTIONS

68. Acknowledgements
Stanley Lab
Dr. Sarah A. Stanley
Dr. Maria Jiménez-González
Rosemary Li
Kavya Devarakonda
Rollie Hampton
Garcia-Ocaña Lab
Dr. Adolfo Garcia-Ocaña
Dr. Carolina Rosselot
Dr. Andrew F Stewart
DIABETES, OBESITY
& METABOLISM
INSTITUTE
Dr. Zhuhao Wu
DEPARTMENT OF
NEUROSCIENCE
THE MICROSCOPY
CORE AND ADVANCED
BIOIMAGING CENTER,
ISMMS
Dr. Deanna Benson
Dr. Nikolaos Tzavaras

69. 1. To learn more about Dr. Sarah Stanley
and Dr. Alexandra Alvarsson’s research,
go to: https://labs.icahn.mssm.edu/stanleylab/
2. To learn more about light sheet microscopy for
high-resolution 3D imaging, go to:
www.miltenyibiotec.com/products/macs-imaging-
and-microscopy/light-sheet-microscopy.html
3. To learn more about
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participating!
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