A Stepwise Laboratory Manual for the Dissection and Illustration of Major Limbic Structures. Evidence from the Klingler’s Technique.

OBJECTIVE: To provide an educational, comprehensive, systematic and stepwise manual for the dissection and illustration of major limbic structures since there is a gap in the pertinent literature. Further, we aim to offer a thorough yet simplied roadmap for laboratory and intraoperative dissections. METHODS: Twenty (20) normal adult, formalin-xed cerebral hemispheres were studied through the ber dissection technique and under the microscope. Stepwise and in tandem medial to lateral and lateral to medial dissections were performed in all specimens aiming to reveal the morphology and spatial relationships of major limbic and paralimbic areas RESULTS: Twelve (12) consecutive, discrete and easily reproducible laboratory anatomical steps are systematically described to reveal the intricate anatomy of the structures of the limbic system. CONCLUSION: Surgical approaches for lesions or functional resections in and around limbic areas pose a challenging task for the neurosurgeon. By employing the ber dissection technique, we were able to provide a stepwise and thorough laboratory guide for the gradual dissection and better comprehension of the morphology and spatial relationships of this specic system. Anatomical manuals like the present study raise interest and enrich anatomical knowledge on complex cerebral areas with the overarching goal to inform surgical practice. cingulate sulcus, sub-parietal sulcus, proximal calcarine ssure, anterior half of the collateral sulcus and rhinal sulcus. The limbic gyrus includes the paraterminal gyri, cingulate gyrus (cingulate pole and cingulate isthmus) and the anterior part of the parahippocampal gyrus is also seen. The subcallosal area (1- highlighted in red color) is seen ventral to the rostrum of the corpus callosum. The uncus is separated from the temporal pole by the shallow rhinal sulcus. B. The subcallosal area is removed to reveal the prehippocampal rudiment, which is seen in the anterior surface of the rostrum and represents the continuation of the indusium griseum (lower left inset). C. The cortex of the cingulate gyrus is removed and the superior arm of the cingulum is shown. The superior surface of the corpus callosum with the overlying indusium griseum (supra-commissural hippocampus) and the medial and lateral longitudinal striae can be appreciated. D. The body of the corpus callosum is removed to access the intraventricular compartment. The gray matter of the medial surface of the thalamus and anterior hypothalamus is peeled away and the mammilothalamic tract of Vicq D’Azyr as well as the pre-commisural and postcommissural part of the fornix are revealed. Inset: Close view of the mammilothalamic tract and the postcommissural fornix. E. The splenium of the corpus callosum is removed and the crus fornix, the posterior part of the dentate gyrus with the fasciola cinerea and the subsplenial gyrus of Andreas Retzius are exhibited. Inset: Close view of the converging conguration of the crus fornix, the dentate gyrus and the subiculum/inferior arm of the cingulum. F. The core of the hemisphere is dissected away and a perpendicular cut is made along the superior margin of the caudate nucleus and at the level of the isthmus of the cingulate gyrus. The hemisphere is turned in the lateral view and the cortex along with the supercial U-bers of the peri-insular area are removed. The supercial anatomy of the insula and the bers of the sagittal stratum are revealed. G. Medial view of the same specimen. The medial parahippocampal cortex is removed while sparing the superior parahippocampal cortex. The inferior arm of the cingulum and the subiculum can be appreciated. H. The arachnoid membrane of the crural and ambient cisterns is removed to detach the medial temporal lobe and the posteromedial orbital lobule from the cerebral peduncle. This maneuver exposes the mbria and the anterior part of the dentate gyrus.. I. A No15 blade is used to make a longitudinal cut along the white matter of the temporal step, 5mm ventral to inferior limiting sulcus. The temporal horn of the lateral ventricle is entered and the alveus of the hippocampus is demonstrated. J. Medial view of the same specimen. The fornix is dissected free and the anatomy of the stria terminalis thalami and choroidal ssure is appreciated. K. Lateral view. The ependymal layer of the roof and tip of the temporal horn is removed to reveal the amygdala. L-M. The anterior part of the parahippocampal gyrus with the hippocampus are dissected away from the hemisphere. For this purpose, a No10 blade is used to cut the anterior part of the temporal stem below the level of the amygdala. In this way, the hippocampus and subiculum and the adjacent structures including the collateral eminence, the uncal, ambient and dentate gyri can be observed in a medial to lateral (M) and lateral to medial(L) view. Lower Left Inset: Close view of the spatial relationships of the head of the hippocampus with respect to the amygdala. N-O. In the last dissection step, the hippocampus is dissected free from the parahippocampal gyrus along


Introduction
The anatomical term "limbic lobe" (latin limbus meaning edge, border), was coined in 1878 by Paul Broca in his essay "Anatomie compare des circonvolutions cerebrales: Le grand lobe limbique et la scissure limbique dans la serie de mammiferes". [2] The introduction of the "great limbic lobe", which marks a natural border between the cerebral hemisphere and diencephalon, would have an enormous impact on neuroanatomical literature for more than a century. From Broca's functional perspective, the limbic lobe represented an archaic structure primarily associated with olfaction in contrast to the high-order lobes linked to cognition, the latter prevailing over the atrophying limbic lobe during evolution. The term limbic lobe (lobus limbicus) is preserved to date (Terminologia Anatomica and Terminologia Neuoanatomica) to de ne a fth cerebral lobe incorporating various anatomical structures. [4,6] Broca's legacy was followed by the pivotal experimental work and clinical observations of researchers like Christr ed Jacob, James Papez and Ivan Yakovlev that would culminate in Paul Maclean's proposal for a discrete and integrated anatomo-functional system underlying our very emotional existence -the limbic system-the paleomammalian element of his triune brain theoretical conception. [3,12,16,14] Since Maclean's proposal, the limbic system, one of the most pervasive models in modern neuroscience, has been extensively expanded and revised to incorporate distinct circuits, believed to subserve affective, mnemonic and behavioural functions. [3,15,20,16] Despite the ongoing effort to elucidate the intricate anatomo-functional architecture and axonal connectivity of this particular network, indeed it seems that there is a paucity in the pertinent literature with regard to its microsurgical anatomy, especially from an educational standpoint. Only a limited number of cadaveric studies have previously utilized the ber dissection technique to elucidate the anatomy of the Jacob/Papez circuit. [17,1,8] In this context, our purpose is to offer for the rst time a comprehensive, systematic and stepwise manual for the dissection and illustration of the limbic lobe and additional major limbic and para-limbic structures through the white matter dissection technique. This recently revitalised method has been incorporated in the neuro-anatomical and neurosurgical education as a valuable tool to acquire a three-dimensional perception of the topographical anatomy and axonal connectivity of the human cerebrum. Hence the present manual aims to provide a valuable resource for the better understanding of the spatial relationships of major limbic structures and assist the neurosurgeon in the mental grasping of the intricate regional anatomy encountered in the operating room.

Materials And Methods
Twenty (20) adult, formalin xed cerebral hemispheres with no gross evidence of disease were treated with the Klingler's preparation (freeze-thaw process). [5,10] All specimens were subsequently investigated through the ber dissection technique with the aid of a microscope (OPMI Plus, Carl Zeiss) and by using surgical micro-instruments including microscissors, micro-forceps and Pen eld micro-dissectors.
The surface anatomy of the medial aspect of the hemisphere was initially observed and recorded to identify the main anatomical landmarks of the limbic lobe and major para-limbic structures according to the Terminologia Anatomica and Terminologia Neuroanatomica. Focused white matter dissections were then carried out in a reproducible medio-lateral and latero-medial stepwise manner with the goal to offer a concrete and thorough three-dimensional perception of the complex regional anatomical relationships.
Each dissection was built upon the idea of gradually revealing and identifying 6 main structures/areas: 1) The limbic ssure 2) The Limbic Gyrus and underlying cingulum 3) The structures of the intralimbic gyrus 4) The insular region 5) The hippocampal-forniceal complex and the mammilo-thalamic tract and 6) The amygdala-caudate-stria terminalis complex. Multiple photos from different angles were obtained in each dissection step to document and vividly illustrate the regional anatomy and spatial relationships of the limbic structures.

Results
Twelve (12) consecutive laboratory anatomical steps are systematically described to reveal the major structures of the limbic system. These steps derive from both medial to lateral and lateral to medial dissections and are summarized in Fig. 1 and Table 1. In the rst step the cortex of the subrostral area (including the paraterminal and paraolfactory gyri) is carefully removed to reveal the prehippocampal rudiment. The prehippocampal rudiment, which corresponds to the precommissural hippocampus, is a thin lamina of gray matter located between the paraterminal gyrus and the lamina terminalis and represents the anterior continuation of the indusium griseum. (Fig. 1B, Fig. 2B)

STEP 2
The cortex of the cingulate gyrus is removed to reveal the superior arm of the cingulum, which typically extends from the subrostral area (pole of the cingulate gyrus) up to the level of the isthmus of the cingulate gyrus. In this step, the indusium griseum -also known as the supra-commissural hippocampusand the longitudinal striae are exposed over the superior surface of the Corpus Callosum (CC). (Fig. 1C, The procedure continues with the core of the hemisphere being dissected free to illustrate more vividly the anatomy of the limbic system. A No10 blade is used to cut along at the level of the superior margin of the caudate nucleus superiorly, the isthmus posteriorly, the cerebral peduncle inferiorly and the posteromedial orbital lobule anteriorly. Then, moving in a lateral to medial direction, the cortex and super cial U-bers of the peri-insular region are carefully removed. The nal product of this step consists of the insular region enfolded within the temporal and frontoparietal opercula seen on the lateral aspect and the thalamus, fornix, parahippocampal gyrus/uncus identi ed on the medial aspect. (Fig. 1F, Fig. 3A,D) STEP 6 Upon removing the cortex of the medial aspect of the parahippocampal gyrus we expose the inferior arm of the cingulum seen to terminate in the entorhinal cortex. The cortex of the intraventricular aspect of the parahippocampal gyrus -known as the subiculum -is left intact. (Fig. 1G, Fig. 3B&C)

STEP 7
The arachnoid membrane of the basal cisterns is carefully removed with micro-forceps so as to dissect the uncus free from the cerebral peduncle and the posteromedial orbital lobule. In this way a thorough look in the topographical anatomy of the mbria and dentate gyrus is allowed. (Fig. 1H, Fig. 3C) STEP 8 Turning the specimen on the lateral side we proceed by removing the cortex and U-Fibers of the superior and middle temporal gyri to expose the inferior limiting sulcus and the temporal stem. Further, we cut with a No15 blade the white matter of the temporal stem approximately 5mm inferior to the inferior limiting sulcus. In this way we enter he temporal horn of the lateral ventricle and identify the choroid plexus, the alveus of the hippocampus and the collateral eminence. (Fig. 1I, Fig. 3E&F) STEP 9 The fornix is gradually dissected off the thalamus starting from the level of the anterior commissure and up to the level of the mbria thus revealing the anatomy of the choroidal ssure. In addition, the stria terminalis connecting the amygdala to the septal nuclei and anterior hypothalamus is vividly illustrated. (Fig. 1K, Fig. 4A)

STEP 10
Again, coming from a lateral-to-medial direction and upon removing the ependymal layer at the level of the tip of the temporal horn, the grey matter of the amygdala becomes evident lying over the anterior part of the temporal stem. The stria terminalis and the tail of the caudate nucleus can be seen terminating at the amygdalae region. (Fig. 1K, Fig. 4B&C)

STEP 11
The most anterior part of the parahippocampal gyrus along with the hippocampus are dissected free from the rest of the specimen. A No10 blade is used to cut the anterior part of the temporal stem at the level of the amygdala. At the end of this step, the parahippocampal gyrus/hippocampal complex can be studied from different angles. From a superolateral view, the head, body and tail of the hippocampus are seen. The alveus, corresponding to the intraventricular surface of the hippocampus as well as the mbria representing the continuation of the fornix can be readily identi ed. Additionally, parts of the parahippocampal gyrus including the collateral eminence at the oor of the temporal horn as well as the subiculum representing the superior aspect of the parahippocampal gyrus in which the hippocampus lies can be observed. From a medial view the hippocampal sulcus, the uncus with its different parts (i.e. uncal and ambient gyri), the subiculum and the dentate gyrus are appreciated. (Fig. 1L&M, Fig. 5)

STEP 12
In the last step, the hippocampus is dissected free from the parahippocampal gyrus along the hippocampal sulcus. Upon completing this step, the structure of the hippocampus can be thoroughly studied. In the medial surface the dentate gyrus demarcated superiorly by the mbrio-dentate sulcus and the inferiorly by hippocampal sulcus inferiorly is illustrated. The different parts of the dentate gyrus including the margo denticularis anteriorly and the fasciola cinerea and gyrus fasciolaris posteriorly are evident. (Fig. 1N&O, Fig. 5D)

Discussion
The ber dissection technique has emerged as an invaluable tool to unravel the ne spatial relationships and axonal connectivity of complex cerebral territories. [19,7,18] This method has evolved during the last 70 years and has been recently employed as a gold-standard procedure for the exploration, illustration and better understanding of the human brain anatomy and connectivity alongside with novel tractographic techniques. In addition to its theoretical signi cance, it also serves as a "navigation tool" for the neurosurgeon by revealing useful 3-dimensional information that can be extrapolated to real-time operative settings mainly in the eld of neuro-oncology, brain mapping and epilepsy.
Numerous studies have previously focused on the gross as well as microscopic anatomy and functional role of the limbic system. Nonetheless, most of the existing literature relies on classical 2-dimensional and fragmentary anatomical illustrations that can be di cult to decipher and translate into the 3dimensional level. Very few authors indeed have used the white matter dissection technique to reveal the structures typically described under the umbrella term "limbic". [1,17,8] Here, we provide a systematic guide for the dissection and illustration of major limbic and paralimbic structures. By dividing the dissection process into 12 distinctive and consecutive steps we aim to offer a simpli ed yet comprehensive approach to understand the highly complex topographic anatomy of this area both in the context of an anatomy laboratory and during real operative scenarios. As anatomical experiments heavily depend on the operator's experience and usually lack reproducibility, stepwise anatomy manuals may compensate for these factors and signi cantly increase the credibility of ndings in a laboratory context. [11] To our knowledge this is the rst attempt to offer a stepwise manual for the dissection of the limbic lobe that can be employed as an educational supplement for both novice and experienced anatomists as well as neurosurgeons.
Challenges in the surgery of limbic and paralimbic areas and the value of anatomy laboratory manuals in modern neurosurgery.
Surgical treatment of lesions or functional resections for epilepsy in or around the limbic system pose a distinct challenge for the neurosurgeon. The deep location, the vicinity to critical neurovascular structures, the complex regional anatomy, the eloquence of the involved cortico-subcortical areas and the ill-de ned anatomical borders make visibility, surgical maneuverability and effective intraoperative dissection arduous. Surgery of insular and peri-insular regions, amygdala and hippocampus, parahippocampal gyrus, cingulate isthmus and cingulate gyrus -areas that in essence make up what is known as the limbic system-requires not only awless surgical skills and optimal bimanual dexterity but a profound, thorough and detailed knowledge of the regional operative anatomy in each case. [9,13] A distinction however has to be made between "static" anatomy, operative anatomy and intraoperative anatomy. The rst entity is mainly conveyed through university anatomical lectures and texts. The second is mastered through dedicated and subspecialized laboratory work and the latter one, which is what the neurosurgeon actually has to face and decipher in the theatre and which is greatly in uenced and usually distorted by the characteristics of the lesion, is patiently learned in real operative settings. Undoubtedly, there is and has to be a linear and progressive relationship between these three entities and the surgeon has to gradually develop from one to the other in order to achieve surgical nesse and mastery. We are in an era where surgery is not regarded as a mere manual technique that is elegantly transmitted from the master to the students by submission and sermon, but has evolved into a proper scienti c specialty. The renowned doctrine "see one, do one, teach one" that conveys the notion of a "con dence based" neurosurgical practice belongs to the past. Novel operative techniques and approaches derive from robust scienti c evidence and original laboratory investigations and propagate through a safe, effective and reproducible intra-operative implementation. This fact documents and supports the concept of what we call "evidence based" surgery. To this end, focused anatomy manuals like the current study provide a simpli ed yet thorough guide that enriches anatomical knowledge, raises interest and awareness on speci c cerebral areas and can act as a roadmap for laboratory or intraoperative dissections.

Conclusion
Limbic and paralimbic structures exhibit a complicated anatomical architecture and therefore surgical approaches targeting lesions or functional resections in this area pose a distinct challenge. Here we provide for the rst time in the pertinent literature a focused, stepwise laboratory manual for the gradual dissection and better comprehension of the subcomponents of this speci c system. Our aim is to provide the novice and experienced anatomist and neurosurgeon with a thorough but simpli ed roadmap for laboratory and intraoperative dissections.

Declarations Disclosures
No funding was received for this study. The authors report no con ict of interest regarding the materials or methods used in this study or the ndings speci ed in this paper. Informed Consent This is a cadaveric study not involving patients or patient related information. All cadaveric specimens used in the current study were obtained by the providing company after strict selfconsent or consent from the legally authorized representatives or next of kin of the donors Consent for publication Not applicable   Step 1: The cortex of the subcallosal area is removed to reveal the prehippocampal rudiment, which lies in the anterior surface of the lamina terminalis. C.
Step 2: The cortex of the cingulate gyrus is peeled away and the superior arm of the cingulum, the superior aspect of the corpus callosum and overlying indusium griseum can be observed. The mammilothalamic tract and the post-commissural fornix are also exposed. D.
Step 3: After removing the splenium of the corpus callosum, three major limbic structures i.e. the crus fornix (yellow color), the dentate gyrus and the inferior arm of the cingulum (red color) can be appreciated. Inset (middle): The location of the prehippocampal rudiment, indusium griseum and subcallosal gyrus is illustrated.  Steps 9-10 of the dissection process A. The fornix is dissected free from the thalamus. The choroidal ssure and stria terminalis thalami are seen (INSET). B. The ependymal layer of the temporal horn is removed. The amygdala can be identi ed at the level of the tip of the temporal horn. Inset: The hippocampus and mbria are illustrated in blue and yellow color respectively. C. Basal view of the specimen: The tail of the caudate nucleus and the stria terminalis thalami, can be seen to terminate in the area of the amygdala. The interrupted line, represents the level of the anterior part of the temporal stem. Anatomy of the intra-limbic gyrus in a right hemisphere The different parts of the intra-limbic gyrus are identi ed i.e. the dentate gyrus also known as the sub-commissural hippocampus, the indusium griseum or supra-commissural hippocampus and the prehippocampal rudiment or precommissural hippocampus.