**2. Morphological organization of NIC in the mammalian pancreas**

These structures consist of autonomic nerve cells, islet cells and nerve fibers in juxtaposition with each other, as described for the first time by Van Campenhout in studies on the histogenesis of islets in human, sheep and dog [24]. Later, Simard [25] confirmed the presence of such complexes in the human pancreas at different ages and termed these structures the neuroinsular complex (NIC) (cited from [26]).

In 1959, Fujita described two types of NIC, which he observed in the fetal and adult pancreas of the dog, cat and rabbit [26]. Some of the ganglia enclosed by the perineural sheath contained islet cells (β- or α-cells) forming NIC type I (NIC I). In NIC I, islet cells contact directly with nerve cells and no intercellular element can be recognized between these two cell types. In NIC type II (NIC II), islet cells lie on the surface of, or even in the midst of, the nerve bundle. However, the distinction between these two types of complexes is conditional because there is an intermediate type of complex in which a mass of islet cells associates with nerve cells in one part of the complex and with nerve fibers in another. Moreover, the ratio between nervous and endocrine elements in different complexes varies greatly [26]. As a variation of NIC I, complexes in which a single or a few nerve cells lying in a corner of a pancreatic islet were observed in the pancreas of the rabbit [26].

Consequently, the structure of the NIC has been intensively studied using histochemical, immunohistochemical methods and electron microscopy [13, 14, 27–31]. Analysis of the thin structure of NIC I and NIC II has shown that endocrine cells contact either directly with axons or with the processes of glial cells (Schwann cells or satellite cells) [27–29]. The gap between the plasmalemma of endocrine cells and glial cells is about 30 nm [28, 29]. Desmosome-like contacts and synaptic contacts between endocrine cells and glial cells or axons have occasionally been found by some authors [28]. As in histological studies, various morphological forms of the NIC were detected using electron microscopy. In many mammals, pancreatic islets are richly innervated by thin nerve fibers, which are located at the periphery of islets, forming periinsular nerve plexuses; they occasionally pass through islets separately or along capillaries [14, 16, 27, 29, 32, 33]. The density of the peri-insular neural network varies between species [27]. It has also been shown that various transitional forms between the classical NIC II in which endocrine cells are located inside nerve bundles and innervated islets are present in the pancreas of the dog [27]. Similarly, various NIC I representing all transitional forms between pure islets with a single neuron and pure ganglia containing only a few endocrine cells have been detected in the pancreas of the cat [28]. Based on these findings, Böck [28] in 1986 introduced the classification of NIC reflecting a gradual transformation between pure neural and pure endocrine structures: 1. An autonomic ganglion with no endocrine cells; 2. NIC I: a) a few endocrine cells in the ganglion; b) a few ganglion cells in the islet; 3. NIC II: a) a single or few endocrine cells integrated with a bundle of nerve fibers; b) heavily innervated islet tissue; 4. A classical islet of Langerhans, either a) innervated or b) not innervated.

In addition to neurons and nerve fibers, glial cells immunopositive for S100 protein and glial fibrillary acidic protein (GFAP) have been detected at the periphery of islets in many mammals [3, 13, 14, 32, 34]. These cells have a triangular or spindle-like shape and possess long, thin leaf-like processes which cover endocrine cells, separating them from the connective tissue and from the acini.

Thus, the NIC are highly organized structures composed of endocrine cells, neurons, nerve fibers and glial cells (Schwann cells or satellite cells). The morphological organization of NIC varies considerably depending on the types of cells forming the complex and their ratios.

#### **3. Structure of NIC in human pancreas**

have been identified in the mouse pancreas [14]. Similar data were obtained in studies on the

Since the classical study by Claude Bernard, which showed that an injury to the floor of the fourth cerebral ventricle caused hyperglycemia, the involvement of the nervous system in the regulation of pancreatic endocrine function and metabolic control has been shown in many studies [17–19]. At the same time, the precise innervation patterns of islets were unknown, particularly in humans [9]. Single nerve cells and nerve ganglia, as myelinated and demyelinated nerve fibers, have been identified in the human pancreas [1, 20–22]. However, the literature data indicate poor innervation of adult human pancreatic islets in comparison with

One of the most interesting features of the mammalian pancreas is that endocrine cells may form highly organized complexes with structures of the nervous system, so-called neuroinsular complexes (NIC). The structure of NIC in the human pancreas has not been studied in

In this chapter, we summarize the literature data and our previous results concerning the morphological organization of NIC in the human fetal and adult pancreas. We also discuss the possible role of the close integration between the nervous system and endocrine cells in the

These structures consist of autonomic nerve cells, islet cells and nerve fibers in juxtaposition with each other, as described for the first time by Van Campenhout in studies on the histogenesis of islets in human, sheep and dog [24]. Later, Simard [25] confirmed the presence of such complexes in the human pancreas at different ages and termed these structures the neuro-

In 1959, Fujita described two types of NIC, which he observed in the fetal and adult pancreas of the dog, cat and rabbit [26]. Some of the ganglia enclosed by the perineural sheath contained islet cells (β- or α-cells) forming NIC type I (NIC I). In NIC I, islet cells contact directly with nerve cells and no intercellular element can be recognized between these two cell types. In NIC type II (NIC II), islet cells lie on the surface of, or even in the midst of, the nerve bundle. However, the distinction between these two types of complexes is conditional because there is an intermediate type of complex in which a mass of islet cells associates with nerve cells in one part of the complex and with nerve fibers in another. Moreover, the ratio between nervous and endocrine elements in different complexes varies greatly [26]. As a variation of NIC I, complexes in which a single or a few nerve cells lying in a corner of a pancreatic islet were

Consequently, the structure of the NIC has been intensively studied using histochemical, immunohistochemical methods and electron microscopy [13, 14, 27–31]. Analysis of the thin structure of NIC I and NIC II has shown that endocrine cells contact either directly with axons

detail since their first description by Van Campenhout [24] and Simard [25].

**2. Morphological organization of NIC in the mammalian pancreas**

pancreas of the rat [15] and nutria [16].

4 Challenges in Pancreatic Pathology

development of the endocrine pancreas.

insular complex (NIC) (cited from [26]).

observed in the pancreas of the rabbit [26].

rodents [1, 9, 20, 23].

According to the literature, few nerve fibers are found in pancreatic islets in adult humans [1, 9, 20, 23]. However, the human pancreas receives extensive innervation, with peculiar growth dynamics during prenatal development [21]. In our previous study,rich innervation of human fetal islets was reported, and both NIC I and NIC II were detected [22].

As mentioned above, the structure of NIC in the human pancreas has not been studied in detail since their first description by Van Campenhout [24] and Simard [25]. In our studies, we investigated the structure of NIC in the human pancreas using immunohistochemistry [22, 35]. We analyzed pancreatic autopsies from 46 fetuses (from the 10th to 40th gestational week (g.w.)), 2 children (3 months old and 3 years old) and 15 adults (24–91 years old). The gestational age of fetuses was determined as the time since the last menstrual period on the basis of the measured crown-rump length and biparietal diameter by ultrasonography. Fetal pancreatic autopsies were divided into four groups, according to the classification of the fetal period [36]: pre-fetal period (10–12 g.w.), early fetal period (13–20 g.w.), middle fetal period (21–28 g.w.) and late fetal period (29–40 g.w.).

To identify structures of the nervous system, we used various neural markers, such as neural cell adhesion molecule (NCAM), peripherin, neuron-specific class III β-tubulin, synaptosomalassociated protein of 25 kDa (SNAP-25), S100 protein and neuron-specific enolase (NSE) [22, 35]. Both types of NIC representing groups of islet cells integrated with ganglionic neurons (NIC I) or with nerve bundles (NIC II) were detected in the fetal pancreas from 14th g.w. onwards [22, 35]. In the pre-fetal period (10–13 g.w.), only contacts between single endocrine cells or small groups and thin nerve fibers were detected [35], and classical NIC I and NIC II were not found.

**Figure 1.** Various forms of NIC I in the human fetal pancreas: single (A–C) or few (D–F) β-cells in the ganglion; pancreatic islets associated with the ganglia (G–L) and few S100-positive cells in the large islet (M–O). Immunofluorescent labeling with antibodies to insulin or glucagon (green) and S100 protein (red).

To identify various subtypes of NIC in the human pancreas we used double immunohistochemical labeling with antibodies to neural makers (S100 protein or NSE) and endocrine hormones (insulin or glucagon) [35]. During prenatal development, i.e. from the 14th to 40th g.w., NIC I was present in the following forms: single ( **Figure 1 A–C** ) or few ( **Figure 1 D–F** ) endocrine cells located among ganglionic cells, pancreatic ganglia associated with islets ( **Figure 1 G–L** ) and few ganglionic cells located at the periphery of islets ( **Figure 1 M-O** ). We also detected various forms of NIC II: single or few endocrine cells in nerve bundles ( **Figure 2 A–C** ), pancreatic islets associated with nerve bundles ( **Figure 2 D–F** ), thin nerve fibers in close proximity to single endocrine cells or small groups and to islets ( **Figure 2 G–L** ).

pre-fetal period (10–12 g.w.), early fetal period (13–20 g.w.), middle fetal period (21–28 g.w.)

To identify structures of the nervous system, we used various neural markers, such as neural cell adhesion molecule (NCAM), peripherin, neuron-specific class III β-tubulin, synaptosomalassociated protein of 25 kDa (SNAP-25), S100 protein and neuron-specific enolase (NSE) [22, 35]. Both types of NIC representing groups of islet cells integrated with ganglionic neurons (NIC I) or with nerve bundles (NIC II) were detected in the fetal pancreas from 14th g.w. onwards [22, 35]. In the pre-fetal period (10–13 g.w.), only contacts between single endocrine cells or small groups and thin nerve fibers were detected [35], and classical NIC I and NIC II

**Figure 1.** Various forms of NIC I in the human fetal pancreas: single (A–C) or few (D–F) β-cells in the ganglion; pancreatic islets associated with the ganglia (G–L) and few S100-positive cells in the large islet (M–O). Immunofluorescent

labeling with antibodies to insulin or glucagon (green) and S100 protein (red).

and late fetal period (29–40 g.w.).

6 Challenges in Pancreatic Pathology

were not found.

**Figure 2.** Various forms of NIC II in the human fetal pancreas: two β-cells in the nerve bundle (A–C); pancreatic islet associated with the nerve bundle (D–F) and thin nerve fibers in close proximity to the islets and single endocrine cells (G–L). Immunofluorescent labeling with antibodies to insulin or glucagon (green) and S100 protein (red).

Thus, the various forms of NIC that we observed in the human fetal pancreas are similar in their morphological organization to the NIC, which were found in the fetal and adult pancreas of other mammals [26–29].

The amount of NIC gradually decreases at birth. In the pancreas of children and adults, NIC are less abundant than in the fetal pancreas [22]. Our quantitative data indicate that the largest number of NIC I was observed in the early and middle fetal periods, during the active morphogenesis of pancreatic islets, whereas at birth (in the late fetal period) and in the adult, NIC II became more prevalent [35]. It should also be noted that NIC I and NIC II in which a single or few endocrine cells were located inside ganglia or in nerve bundles were found only in the fetal pancreas. We did not find these types of NIC in the adult pancreas, probably due to an insufficient number of fields of observation. Therefore, we could not exclude that these types of NIC can be present in the adult pancreas, but they are rare. NIC I in which pancreatic islets were associated with ganglia were more numerous in the fetal pancreas and were occasionally found in the pancreas of children [22] and adults [35]. Among the NIC II, at all investigated stages of development, as well as in children and adults, interactions between thin nerve fibers and endocrine cells located separately or inside the islets prevailed [35].

To identify whether glial (Schwann) cells cover the periphery of islets in humans, as in other mammals, we used immunohistochemical labeling with antibodies against S100 protein and GFAP as well as electron microscopy. We found small S100-positive cells with thin processes at the periphery of some islets in humans [37, 38]. The same small oval, triangular or elongated cells with long thin processes were observed in the fetal pancreas using electron microscopy [38]. The processes of these cells were often cover or surround nerve fibers passing into islets [38]. In contrast to mice and rats [3, 13], these cells were immunonegative to GFAP. However, according to their ultrastructural characteristics and integration with nerve fibers, these small S100-positive cells with thin processes that we detected in the human pancreas correspond to the glial (Schwann) cells observed at the periphery of islets in other mammals [3, 13, 14, 34]. It should be noted that, in humans, S100-positive glial cells are present only in some islets in small numbers and their processes do not cover endocrine cells, as has been described in other mammals [3, 13, 14, 32, 34].

Taken together, our findings indicate that, in the human pancreas, NIC are more abundant and variable in their morphological organization in the prenatal period, i.e. during the active morphogenesis of pancreatic islets. Based on these findings, we suggest that the nervous system may be involved in the development of the human endocrine pancreas. In the next part of this chapter, we discuss the existing points of view on the possible functional role of NIC.
