**2. Relationship between perivascular space (PVS) and keratin-negative and/or epithelium-free areas**

In the medulla, the perivascular space (PVS) [7, 9–12]—that is, the dilated primary septum (PS)—carries the blood vessels, which locate "outside" the basal lamina of the thymus [1, 2, 12, 13], but Foxn-1, which is a thymic epithelial cell-specific

**6**

*Thymus*

**References**

[1] Sadler TW. Langman's Medical Embryology. Philadelphia Pa London: Lippincott Williams & Wilkins; 2011

of thymus organogenesis and morphogenesis. Development. 2011;**138**(18):3865-3878

[2] Gordon J, Manley NR. Mechanisms

Current Opinion in Immunology.

thymic function: Then and now. Cytokine. 2019;**120**:202-209

Reviews. 2016;**271**(1):56-71

[12] El-Kadiry AE-H, Rafei M. Restoring

[13] Chaudhry MS et al. Thymus: The next (re)generation. Immunological

[14] Fan Y et al. Bioengineering thymus organoids to restore thymic function and induce donor-specific immune tolerance to allografts. Molecular Therapy. 2015;**23**(7):1262-1277

2013;**25**(4):516-522

[3] Larsen WJ. Human Embryology. Churchill Livingstone; 2001. Available from: https://www.elsevier.com/ books/larsens-human-embryology/ schoenwolf/978-1-4557-0684-6

[4] Pierpaoli W, Besedovsky HO. Role of the thymus in programming of neuroendocrine functions. Clinical and Experimental Immunology.

[5] Mathis D, Benoist C. Back to central tolerance. Immunity.

[6] Takaba H, Takayanagi H. The mechanisms of T cell selection in the thymus. Trends in Immunology.

[7] Csaba G. The immunoendocrine thymus as a pacemaker of lifespan. Acta Microbiologica et Immunologica Hungarica. 2016;**63**(2):139-158

[8] Nunes-Alves C et al. Tolerance has its limits: How the thymus copes with infection. Trends in Immunology.

[9] Calder AE et al. Thymic involution:

immunology. Neuroimmunomodulation.

Where endocrinology meets

[10] Palmer D. The effect of age on thymic function. Frontiers in

[11] Ventevogel MS, Sempowski GD. Thymic rejuvenation and aging.

Immunology. 2013;**4**:316

1975;**20**(2):323

2004;**20**(5):509-516

2017;**38**(11):805-816

2013;**34**(10):502-510

2011;**18**(5):281-289

#### **Figure 1.**

*Double staining of chicken thymus: cytokeratin (green) and laminin (red). At the PS, the laminin shows a continuous basal lamina, which becomes discontinuous in the EFA. The KNA/PVS receives the blood vessels.*

transcription factor [14], regulates thymic vascularization [15]. At the corticomedullary junction, PS merges to the PVS of the medulla [9, 10]. A continuous basal lamina covers the thymic parenchyma toward the capsule and PS, and it becomes discontinuous at the end of the PS (**Figure 1**) [2, 7] where it turns to be the PVS or KNA/ EFA. In the thymic medulla, the lack of blood-thymus barrier [16] may be explained by the discontinuity of the basal lamina on the border of the KNA/EFA [2]. The discontinuity of the basal lamina suggests that the microenvironment of the KNA/ EFA and PVS is identical. Silver impregnation shows that both the PS and the KNA/ EFA consist of reticular connective tissue [2, 9, 17] and have common extracellular matrix [11]. Secondary septae appear after formation of the cortex and medulla, and they are just small invaginations of the capsule and usually do not reach the medulla and do not receive blood vessels. The medullary EFA occupies about one-fifth of the rat thymus [18]. In chicken our morphometric studies confirm the considerable size of the KNA, that is, close to 50% of the medulla [2]. The border of the keratin-positive network (KPN) and KNA is an epithelial-mesenchymal border that could be the functional cortico-medullary (CM) border of the thymus [2]. The KPN-KNA/EFA border is supported by cellular background, unlike the hematoxylin-eosin-stained, classical CM border, which is based only on lymphocyte density and subsequently stainability. The mesenchymal tissue of the PVS develops from neural crest cells [19–22]. The PVS is a transit zone of migratory cells between the thymus and circulation [12, 23].

Anti-cytokeratin immunostaining identifies the KPN and KNA/EFA in both embryonic and postembryonic chicken thymuses. In an 11-day-old chicken embryo, the thymic epithelial anlage shows a starfish-shaped form (**Figure 2**). Between the 5–6 secondary epithelial cords, the unstained PS(s) consist of mesenchyme. During the next two ED (11 and 13), the cortical epithelial cells rapidly proliferate resulting in enlarged thymic rudiment (**Figure 3**) which is colonized by hematopoietic cells. In 11-ED-old birds, the wide PS became narrow, and the bottom of the PS is involved into the medulla as the KNA/EFA.

The PS is going on as the KNA/EFA, and both regions consist of reticular connective tissue stained with silver impregnation [2, 6, 17, 24]. Mesenchymal markers desmin and ER-TR7 [6, 25] revealed specific staining in the capsule, septae, and medullary PVS. In the PVS, the neural crest cells differentiate into smooth muscle cells of thymic blood vessels and pericytes of thymic capillaries [21]. These histological findings suggest the common origin of the PS and KNA/EFA: namely, the KNA/EFA develops from the cranial neural crest cells [19, 21, 26].

**9**

**Figure 2.**

**Figure 3.**

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses*

**3. Keratin-negative area/epithelium-free area**

*bottom of the PS (arrowhead) dilates and becomes the KNA/EFA of medulla (star).*

is located in close association with the blood vessels [2].

Mesenchymal cells and fibroblasts express vimentin intermediate filament. Cortical thymocytes and epithelial cells are vimentin negative (**Figure4**), but thymic medulla shows homogenous staining pattern, indicating that keratin-positive and keratinnegative compartments cannot be distinguished (**Figure 5**). The homogenous vimentin staining of the medulla may indicate that the medullary epithelial cells express vimentin intermediate filament. Hassall's bodies are vimentin negative (**Figure 5**), like cortical epithelial cells. In the majority of vimentin-positive cells, the immunoreaction appears in the periphery of the cell cytoplasm. The nature of the medullary vimentin-positive cells is not clear, because vimentin can colocalize with other intermediate filaments, like neurofilament, cytokeratin, and desmin; therefore the anti-vimentin immunostaining used for identification of mesenchymal cells is limited [25]. Blood vessels and dendritic cells are the most significant structures of the KNA/EFA. Anti-von Willebrand factor identifies endothelial cells (**Figure6**). Transmission electron microscopy shows the organelle-rich cytoplasm of the interdigitating dendritic cell (IDC) (**Figure 7**). The IDC

*13 ED chicken thymus: the rapid proliferation of epithelial cells enlarged the thymic cortical epithelium. The* 

*11 ED chicken thymus: anti-cytokeratin. The starfish-shaped epithelial thymic anlage shows the branching of* 

*the primary epithelial cord to 5–6 secondary cords. The dashed circle outlines the future medulla.*

*DOI: http://dx.doi.org/10.5772/intechopen.88588*

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses DOI: http://dx.doi.org/10.5772/intechopen.88588*

#### **Figure 2.**

*Thymus*

**Figure 1.**

transcription factor [14], regulates thymic vascularization [15]. At the corticomedullary junction, PS merges to the PVS of the medulla [9, 10]. A continuous basal lamina covers the thymic parenchyma toward the capsule and PS, and it becomes discontinuous at the end of the PS (**Figure 1**) [2, 7] where it turns to be the PVS or KNA/ EFA. In the thymic medulla, the lack of blood-thymus barrier [16] may be explained by the discontinuity of the basal lamina on the border of the KNA/EFA [2]. The discontinuity of the basal lamina suggests that the microenvironment of the KNA/ EFA and PVS is identical. Silver impregnation shows that both the PS and the KNA/ EFA consist of reticular connective tissue [2, 9, 17] and have common extracellular matrix [11]. Secondary septae appear after formation of the cortex and medulla, and they are just small invaginations of the capsule and usually do not reach the medulla and do not receive blood vessels. The medullary EFA occupies about one-fifth of the rat thymus [18]. In chicken our morphometric studies confirm the considerable size of the KNA, that is, close to 50% of the medulla [2]. The border of the keratin-positive network (KPN) and KNA is an epithelial-mesenchymal border that could be the functional cortico-medullary (CM) border of the thymus [2]. The KPN-KNA/EFA border is supported by cellular background, unlike the hematoxylin-eosin-stained, classical CM border, which is based only on lymphocyte density and subsequently stainability. The mesenchymal tissue of the PVS develops from neural crest cells [19–22]. The PVS is a transit zone of migratory cells between the thymus and circulation [12, 23]. Anti-cytokeratin immunostaining identifies the KPN and KNA/EFA in both embryonic and postembryonic chicken thymuses. In an 11-day-old chicken embryo, the thymic epithelial anlage shows a starfish-shaped form (**Figure 2**). Between the 5–6 secondary epithelial cords, the unstained PS(s) consist of mesenchyme. During the next two ED (11 and 13), the cortical epithelial cells rapidly proliferate resulting in enlarged thymic rudiment (**Figure 3**) which is colonized by hematopoietic cells. In 11-ED-old birds, the wide PS became narrow, and the bottom of the PS is

*Double staining of chicken thymus: cytokeratin (green) and laminin (red). At the PS, the laminin shows a continuous basal lamina, which becomes discontinuous in the EFA. The KNA/PVS receives the blood vessels.*

The PS is going on as the KNA/EFA, and both regions consist of reticular connective tissue stained with silver impregnation [2, 6, 17, 24]. Mesenchymal markers desmin and ER-TR7 [6, 25] revealed specific staining in the capsule, septae, and medullary PVS. In the PVS, the neural crest cells differentiate into smooth muscle cells of thymic blood vessels and pericytes of thymic capillaries [21]. These histological findings suggest the common origin of the PS and KNA/EFA: namely, the

KNA/EFA develops from the cranial neural crest cells [19, 21, 26].

**8**

involved into the medulla as the KNA/EFA.

*11 ED chicken thymus: anti-cytokeratin. The starfish-shaped epithelial thymic anlage shows the branching of the primary epithelial cord to 5–6 secondary cords. The dashed circle outlines the future medulla.*

#### **Figure 3.**

*13 ED chicken thymus: the rapid proliferation of epithelial cells enlarged the thymic cortical epithelium. The bottom of the PS (arrowhead) dilates and becomes the KNA/EFA of medulla (star).*
